meiotic gene conversion mutants in saccharomyces ... · cqqright 0 1985 by the genetics society of...

Cqqright 0 1985 by the Genetics Society of Anierica

MEIOTIC GENE CONVERSION M U T A N T S I N SACCHAROMYCES CEREVISIAE. I. ISOLATION AND

CHARACTERIZATION OF P M S l - 1 A N D ~ ~ 4 ~ 1 - 2

MARSHA S. WILLIAMSON,* JOHN C. GAME? AND SEYMOUR FOGEL*

*Department of Genetics, University of Calijiornia, and +Division of Biology and Medicine, Lawrence Berkeley Laboratory, Berkeley, Calfornia 94720

Manuscript received February 3, 1984 Revised copy accepted April 13, 1985

ABSTRACT

The pms l mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PAIS1 were characteri~ed. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic niutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in p m s l homo7ygotes. The PMS1 locus maps near MET4 on chromosome XIV. T h e PMSI gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. T h e PMSZ gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.

0 better understand meiotic recombination, it is important to identify the genes controlling its component reactions. Gene conversion, a ubiquitous

process occurring in both meiotic and mitotic cells, is currently viewed as an integral aspect of the process that generates reciprocal recombination (for reviews see FOGEL et al. 1979; ESPOSITO and WACSTAFF 1981). There are currently two contrasting types of models to account for the mechanism of meiotic recombination. Classical models (HOLLIDAY 1964; HOTCHKISS 1974; MESELSON and RADDINC 1975) postulate that heteroduplex DNA (hDNA) is the basic recombination intermediate. According to these models, 6:2 and 2:6 gene conversions and 4:4 restorations are the products of mismatch correction (via excision-resynthesis). [Note that meiotic tetrad segregation ratios are given as octads (FOCEL et al. 1979); the first and second integers, respectively, denote the number of wild-type and mutant segregants.] On hDNA models, recombination initiation involves single-strand breaks, but the contrasting double-

Genetics 110 609-646 August, 1985

610 M. S . WILLIAMSON, J. C. GAME AND S. FOGEL

strand break repair (DSBR) model developed by SZOSTAK et al. (1983) proposes that recombination initiates via double-strand break formation. 6:2 and 2:6 conversions arise solely from repair in regions containing double-strand gaps, rather than from correction of mismatches in hDNA. These alternative models could represent two different pathways that process meiotic recombination intermediates in the same cell. The present work was undertaken to obtain more information concerning the mechanism(s) of meiotic recombination and the nature of recombination intermediates in yeast. For reviews concerning the genetic phenomenology and the biochemistry of meiotic recombination and gene conversion see STADLER (1 973), HASTINGS (1 975), PUKKILA (1 977), RADDING (1973, 1978, 1982), FOGEL et al. (1979), FOGEL, MORTIMER and LUSNAK (1 98 1, 1983), STAHL (1979) and WHITEHOUSE (1 982).

T o date, much of the genetic evidence for molecular intermediates in the gene conversion process in eukaryotes has been based on the study of meiotic segregation patterns in ascomycetes such as yeast (FOGEL and HURST 1967; FOGEL, MORTIMER and LUSNAK 1981), Sordaria (KITANI and OLIVE 1967, 1969; YU-SUN, WICKRAMARANTE and WHITEHOUSE, 1977), Ascobolus (LEBLON 1972a,b; LEBLON and PAQUETTE 1978; ROSSIGNOL, PAQUETTE and NICOLAS 1979) and Neurospora (CASE and GILES 1958, 1964; STADLER and TOWE 1963). The occurrence of postmeiotic segregation (PMS) in these organisms is considered the most direct genetic evidence for the existence of hDNA intermediates in the gene conversion process. In yeast, such segregations (5:3, 3:5, ab4:4, etc.) were first reported by ESPOSITO (1971), but they had been previously documented in Sordaria (OLIVE 1956; KITANI, OLIVE and EL-ANI 1962) and other fungi. PMS is generally considered to occur when an uncorrected mismatch within hDNA is resolved by replication at the first mitotic division after meiosis. Suppression of PMS at one heterozygous site, in the presence of an adjacent heterozygosity, has been observed at the b2 locus in Ascobolus (LEBLON and ROSSIGNOL 1973; ROSSIGNOL and HAEDENS 1978) and the arg4 (FOGEL et al. 1979; FOGEL, MORTIMER and LUSNAK 1981) and his1 (SAVAGE and HASTINGS 1981; HASTINGS 1984) loci in yeast. This suppression has been postulated to result when two mismatches occur in the same hDNA segment and correction stimulated at or near one mismatch extends to the second mismatch (which is normally less efficiently corrected). The phenomenon suggests that the recombination intermediate sometimes includes hDNA regions spanning intragenic sites. In addition, the patterns of co-conversion that are observed when segregations involving multiple sites within a gene are studied support the hypothesis that recombination intermediates have considerable ex- tent (FOGEL et al. 1979; FOGEL, MORTIMER and LUSNAK 1981; ROSSIGNOL, PAQUETTE and NICOLAS 1979).

Mutants with altered recombination profiles have been isolated in several eukaryotic organisms (for review see BAKER et al. 1976). Studies with these mutants may ultimately prove valuable for defining the molecular details of meiotic recombination. In higher eukaryotes, recent studies with recombination-defective Drosophila mei mutants (CARPENTER 1982, 1984) reveal that gene conversion and reciprocal recombination can be mutationally uncoupled.

YEAST GENE CONVERSION MUTANTS 61 1

Crossing over is severely reduced in these mutants; however, the frequency of gene conversion appears normal or elevated. Among the fungi, natural variants in Neurospora (CATCHESIDE 1975) and Ascobolus (HELMI and LAMB 1979) affect the frequency of recombination initiation. Yeast mutants such as rad50 (GAME et al. 1980) and s p o l l (KLAPHOLZ and ESPOSITO 1980a,b; BRUSCHI and ESPOSITO 1982) abolish meiotic recombination altogether, yielding inviable spores unless the first meiotic division is mutationally bypassed (MALONE and ESPOSITO 1981). A yeast variant (pacl) with complex effects on meiotic recombination, described by DAVIDOW and BYERS ( 1 984), yields enhanced meiotic gene conversion and exchange frequencies following pachytene arrest at 36 O . The PMS frequency increases at some sites, whereas 16:21 segregation frequencies increase at all sites. Pachytene arrest may, therefore, increase the length of the recombination intermediate as well as the recombination initiation frequency. Mutations at several loci in yeast lower meiotic prototroph frequencies. The rec4 mutation (RODARTE-RAMON and MORTIMER 1972; Ro- DARTE-RAMON 1972; SANFILLIPO 1977) has a locus-specific effect and presumably causes heteroduplex length to increase when recombination initiates at or near arg4. The con (ROTH and FOGEL 1971; FOGEL and ROTH 1974) and mei (ROTH 1976) mutants have meiotic hypo-rec phenotypes. Some fail to complete premeiotic DNA synthesis and are, therefore, sporulation deficient. Other mutants sporulate normally, but spore viability is very low, indicating that a general function required for normal chromosome disjunction may be affected.

For this study, a selective system similar to that designed by ROTH and FOGEL (1 97 1 ) was devised for sensitive detection of enhanced, rather than diminished, prototroph frequencies at three heteroallelic sites in a haploid strain disomic for chromosome 111. Both dominant and recessive mutations can be recovered using this system (except on chromosome IZI, where only dominants can be detected). Heterozygosity at MAT allows the disomic strains to undergo premeiotic DNA synthesis, meiotic recombination and incipient sporulation. Un- der sporulation conditions, such haploid cells die after commitment to the meiotic reduction division (ROTH and FOGEL 1971; FOGEL and ROTH 1974). However, in yeast, commitment to recombination and commitment to the meiotic divisions can be separated in time (SHERMAN and ROMAN 1963). Pro- totrophic recombinants are assayed by interrupting meiosis; cells exposed to sporulation conditions are plated back to selective vegetative media. Proto- trophic recombinants that have not yet committed to the meiotic reduction division will survive and commence mitotic growth (ESPOSITO, PLOTKIN and ESPOSITO 1974; KLAPHOLZ and ESPOSITO 1980b). It was expected that selection for a hyper- rather than a hyporecombination phenotype would yield some mutant isolates that were not blocked in meiosis and/or sporulation in the diploid state. A subset of the recovered putative mutants also displayed elevated spontaneous mutation rates and elevated PMS frequencies; these are the subject of the present study. Recombination intermediate processing, e.g., heteroduplex formation or mismatch correction may be altered in these strains. These mutants would be difficult to detect by directly screening for increases in PMS frequency because there is no selective system for monitoring such

612 M. S. WILLIAMSON, J. C. GAME AND S. FOGEL

MW 104-1 B

his4-4 leu2- I MATd thr4- 1 MAL2

his4-519 leu2-27 MATa thr4- 16 me12 Ill , , 17 ‘ . 5 T 2 5 1 22 63

IV t r p l - I C A N I ~ ura3- 1 ade2- 1

FIGURE 1 .--Genotype of M W 1 0 4 - l B , a triply heteroallelic MATa/MATct haploid strain disomic for chromosome I I I . Map distances are given in centimorgans. The order of heteroalleles with respect to the centromere is not implied.

_t_ xv - - - - - - - - v- - - - - ; - d.5 44 8 - 88

rare events. This paper describes the isolation and characterization of strains carrying the two recessive allelic tnutations conferring elevated PMS, designated pmsl -1 and pmsl-2.

MATERIALS AND METHODS

Media: YEPD, GNA, synthetic complete (SC), supplemented sporulation (KAC) and omission media have been described previously (FOCEL and MORTIMER 1970; CAMPBELL, FOCEL and LUSNAK 1975). Omission media are described by abbreviations for the nutrilite(s) they lack. For example, HIS, LEU and THR represent media without histidine, leucine and threonine, respectively. Can- avanine (CAN) medium is an arginine-deficient medium supplemented with 30 mg/liter of L-

canavanine (a toxic arginine analog). Presporulation medium (PSM) is described by KUENZI and ROTH (1974).

Genetic techniques: Standard techniques were used for mass mating, zygote isolation and ascus digestion (MORTIMER and HAWTHORNE 1969). For segregation analyses, the plate dissection method was employed (FOCEL et al. 1979) . For segregation analyses of the mutator phenotype and cosegregation of the meiotic and mitotic mutant phenotypes, 1 2 (rather than 20) tetrads were placed on a single plate. Complementation tests allowing PMS detection were performed according to the method of FOGEL et al. ( 1 979).

Genetic markers and strains: A spontaneous mutation, thr4-16, isolated by the inositol-less death protocol (CULRERTSON and HENRY 1975), and a SUPX” mutation, selected by cosuppression of the his4-4 and leu2-I ochre alleles, were isolated in this laboratory for this study. Mapping strains carrying spol 1 were provided by S. KLAPHOLZ and R. E. ESPOSITO. All other markers and strains were obtained from this laboratory’s stocks. The add-18 allele was isolated by M. ESPOSITO (1968). T h e leu2-27, his4-4, his4-519 alleles and S U F I , a frameshift suppressor of his4-519, were isolated in G. R. Fink’s laboratory (FINK 1964; CULRERTSON et al. 1977). The arg4 alleles, described in detail by FOCEL et al. (1979) and FOCEL and MORTIMER (1971), and the leu2-1 allele were isolated in R. K. Mortinier’s laboratory.

Gene conversion mutants were isolated using the chromosome III disomic (n -t 1) strain, M W 1 0 4 - l B , shown in Figure 1. Genetic map distances (MORTIMER and SCHILD 1980) are given in centinrorgans (cM). The presence of heteroalleles at his4, leu2 and thr4 in the trisomic parent MW104 and in disomic ascosporal colonies was validated by induced mitotic gene conversion to prototrophy after exposure of colony prints to a nonlethal dose (40 J/m2) of UV light from a GE germicidal lamp. The flux MUS measured by a LATARJET (LATARJET, MORENNE and BERCER 1953) ,U\’ dosimeter. M W 1 0 4 - I B was selected for mutagenesis after verifying the presence of heteroalleles at each of the three test loci. The inability of this ascosporal colony to mate or sporulate is consistent with heterozygosity at MAT.

YEAST GENE CONVERSION MUTANTS 613

Isolation ofpms mutants: A stationary phase culture of MW104-1B was "agenized with ethyl rnethanesulfonate (EMS) by the method of LINDECREN et al. (1965). Cells exposed to EMS for 70 min, yielding 25% survival, were diluted and plated onto YEPD. After 5 days of incubation at 30", single colony survivors were transferred with toothpicks to YEPD master plates with 50 colonies/master. Each master plate also contained two untreated samples of the parental strain as controls. After 48 hr of growth, each master was replica plated onto HIS, LEU and T H R single omission media, one CAN plate and one supplemented KAC plate. The latter medium maximizes meiosis induction in slowly sporulating strains (ROTH and FOCEL 1971; FOCEL and ROTH 1974). After 4 days, the single omission media plates were screened for prints with high spontaneous mitotic prototroph backgrounds, and the CAN plates were screened for prints with an abnormally high frequency of CAN-resistant outgrowths (indicating a possible mitotic mutator phenotype). After 2 days the KAC sporulation plate was replica plated directly onto one plate each of HIS, LEU and T H R vegetative media (to assess prototrophic recombinants among cells uncommitted to the meiotic divisions). After 5 days the replica plates were examined for appearance of leucine-, threonine- and histidine-independent colonies of meiotic origin. Putative mutants displaying elevated meiotic prototroph frequencies on any or all omission media were retested at 30" and 34". The frequency of UV-induced prototrophy was also monitored at this time.

Genetic analysis

Isolation of mating segregants: Disomic MATaIMATa or MATaIMATa and haploid MATa or M A T a derivatives of MW 104-1B were efficiently obtained by screening for mating ability (complementation for an adenine requirement) among colonies derived from cells surviving prolonged exposure to KAC (4-7 days), a protocol suggested by M. ESPOSITO (personal communication). Enhanced UV-induced prototrophy identified disomic maters heteroallelic at one or more loci. Mating-type segregation patterns in outcrosses to standard testers distinguished ordinary or euploid haploids from homoallelic disomic haploids.

Strain construction for initial characterization: Diploids were constructed from successive back- crosses of haploids derived from putative mutants and MW104-1B to a haploid strain closely related to S K I , a diploid strain noted for synchronous and high sporulation (KUENZI and ROTH 1974). These strains were homozygous or heterozygous for each putative mutation or homozygous for wild type. Most strains were heteroallelic at his4, leu2 and thr4, and all were CAN sensitive. These strains (or their haploid derivatives) were used to generate data on the segregation of the mutator phenotype, mitotic prototroph rates, UV survival curves, UV-induced prototroph frequencies and meiotic prototroph frequencies.

Scoring the mutator phenotype: T o score the mutator phenotype segregations, ascosporal colony replica prints (5 mm) on CAN media were assessed for the presence of CAN-resistant outgrowths (papillae) after 4 days. On a 12-tetrad grid, wild-type prints typically display two or fewer papillae, whereas mutant prints contain three or more. Unlike the characteristic growth us. nongrowth phenotype used in scoring a nutritional mutant, there is overlap between these frequency-based mutant and wild-type phenotypes. Misclassification occurs 3-5% of the time. The assumption that pmsl strains are similar to wild type with respect to any residual background growth of CAN- sensitive cells and their ability to express CAN resistance on CAN-containing medium is supported by the observation that the frequency of UV-induced papillae on CAN plates is similar in pmsl and wild-type strains. Only tetrads in which all four spore colony prints produced CAN-resistant papillae after exposure to 40 J/m' of UV were scored for spontaneous production of resistant outgrowths.

Cosegregation of mutator and meiotic prototroph phenotypes: Diploids tetrasomic for chromosome I I I , heterozygous for the unlinked pmsl mutation under study, and heteroallelic at h i d , leu2 and thr4 (with two copies of each allele for each locus) were constructed by crossing together MATa/ MATa and MATaIMATa disomes derived from both MW104-1B and the putative mutant strains. Cosegregation of the mutator phenotype (elevated mutation to CAN resistance) and the meiotic phenotype (enhanced prototroph frequency) was monitored in his4-4/his4-519 MATa/MATa ascosporal colonies obtained from dissected tetrads of these diploids. Tetrads with all four spore colonies appropriately marked were rare among total tetrads. To ensure a relatively random sample, no more than two colonies were sampled from any tetrad.


TABLE 1

Allele identtjications based on complementation or suppression (his4-4 + / + his4-519)

Growth on histidineless medium

X his4-519 X his4-4 Spore genotype Alone SUPX” SUFl

- + - 4-4 + + + 4-519

4-4 4-519 + + + + +

- - - - -

Strain construction for conuersion studies: Mutant and wild-type haploid derivatives of MW 104- 1B were crossed to a strain carrying many additional markers for assessing pmsl effects on gene conversion. Most diploid strains carried the following marker configuration:

his4-4 leu2-I MAT@ thr4-1 MAL2 his4-519 LEU2 MATa THR4 mal2

ARG4 T H R l cup13 trpl-1 ADE8 arg4-16 thrl CUPI‘ T R P l a d d - 1 8

HIS2 LYSl MET13 ura3-1 ade2-1 his2 lysl-1 met13 URA3 ade2-1

Appropriately marked mutator segregants from heterozygotes served for construction of pmsl-1 and pms l -2 homozygotes. Because the mutator phenotype (elevated mutation to CAN resistance) could not be directly assessed in arg4 auxotrophs, segregants carrying pmsl and arg4 alleles were identified by outcrossing. Diploids for the a r g l three-point study were constructed by crossing pms l -1 haploids carrying arg4-3 and arg4-36 (identified by outcrossing) to PMSl arg4-16 haploids. The configuration of segregating alleles at arg l was: 4-3 + 4-36/+ 4-16 + (arg4-3 is centromere proximal). Most of these strains carried the additional markers listed above.

Meiotic segregation data collected from unselected tetrads in these multiply marked strains served to characterize the mutant effects on the basic conversion frequency, I 6:2 I frequencies, PMS ( 1 5:3 I ) frequencies and reciprocal exchange (for definitions, see below). Tester systems to detect PMS based on allele suppressibility and/or intragenic complementation of segregating heteroalleles at his4 and arg4 are outlined in Tables 1 and 2. All possible spore colony or sector genotypes are unambiguously identified (see FOGEI. et a l . 1979; FOGEL, MORTIMER and LUSNAK

-----

I98 1).

Quantitative techniques

Mutations rates: Mutation rates to CAN resistance and reversion rates for the ochre suppressible allele, his4-4, were estimated in haploids by the method of LURIA and DELBRUCK (1943) adapted to yeast (FOGEL, LAX and HURST 1978; MALONEY and FOGEL 1980). If we assume that a Poisson distribution of mutant papillae occurs among the prints, the mutation rate = -In P(O)/n cells/ print, where P(0) = the proportion of single-cell colony prints with no mutant outgrowths. When papilla frequencies were low, mutation rates were calculated directly from observed mean frequencies. The rates in these experiments were not corrected for viability and could be underesti- mated if cell viability were lower than 100%.

Spontaneous mitotic prototroph frequencies: The method of the median (LEA and COULSON 1948) was employed. T h e procedure is similar to that described by ESPOSITO (1978) and PRAKASH et al. (1980). Rates were calculated from the following expression (DRAKE 1970):

0.4343 median frequency Rate = (log,, final cells) - (log,, initial cells)

YEAST GENE CONVERSION MUTANTS

TABLE 2

Allele identijcations based on complementation or suppression (arg4-3 + arg4-36 / + arg4-16 +)

615

Growth on arginineless medium

X arg4-3 X arg4-16 Spore genotype Alone SUPX' X arg4-27 SUP??

+ 16 + 3 + 36 3 + + + + 36 3 16 + + 16 36 3 16 36 + + +

For each diploid heteroallelic at h i d , leu2 and thr4, 25 separate 3-ml cultures were used and calculations were corrected for viability.

UV sensitivity and UV-induced gene conversion: Diploid cultures were grown in liquid YEPD to midlog or early stationary phase. Cells were then washed and plated onto SC or omission media and irradiated with a GE germicidal lamp at a dose rate of 34 J/m2/sec or 8.3 J/m2/sec, depending on the experiment. Data from honioallelic diploids were used to determine the prototroph frequency arising froni mutation at each UV dose in heteroallelic diploids. These contributions were subtracted from the observed frequency to obtain the frequency of prototrophs per survivor that arose from recombination.

Meiotic prototroph frequencies: Method 1. Diploid or disomic haploid cells were grown in liquid PSM to late log phase and transferred after washing to 1% potassium acetate + adenine (100 rg / nil) at a density of 2 X IO'/ml. After various intervals in liquid sporulation medium, samples were plated onto appropriate omission media as well as SC to determine prototrophs per survivor by the interrupted meiosis procedure of SHERMAN and ROMAN (1963). (See the Introduction for the descriptive biology relating to meiotic pull-back experiments.) Initial mitotic prototroph frequencies were subtracted from values obtained at various time points. Percent sporulation was also recorded. For diploids, samples contained mostly whole asci and approximately 10-20% unsporulated diploid cells.

Method 2. Diploids sporulated for 48-96 hr were obtained from replica prints on KAC plates and digested with glusulase for 1-2 hr to disrupt unsporulated diploid cells and release spores from ascus walls. The spore suspension was diluted in distilled water and left at room temperature overnight. Spores were then dispersed by mixing with glass beads (0.5 mm diameter) and plated onto omission media as well as SC to determine prototroph frequency per survivor. Premeiotic prototroph background frequencies were not determined for the experiments shown. This latter method estimates the frequency of prototrophs per spore.

Statistical analysis: For mutation rates, 95% confidence intervals were calculated assuming a Poisson distribution of events (CROW and GARDNER 1959). Nonoverlapping distributions are considered significantly different from each other. 2 tests (SOKAL and ROHLF 1969), employed to determine the probability that two proportions belong to the same population of events, were used to compare conversion event class frequencies in mutant and wild type. Z values indicating P 5 0.05 were considered significant. x2 tests were applied to test homogeneity of distributions of exchange ranks and other distributions (e.g., spore survival patterns and distribution o f conversion classes among aberrant segregations or total segregations) between mutant and controls. When sample size for any class was less than 5, G tests (SOKAL and ROHLF 1969) were applied to obtain x2 values. x2 values indicating P 5 0.05 were considered significant.

Abbreviations and definitions: Unless otherwise specified, basic definitions and conventions follow those given by FOCEL et al. (1979). We are here defining gene conversion events as all nonre-

616

ciprocal recombination events that result in aberrant (non-Mendelian) segregations, including PMS. Although cryptic conversion events resulting in 4:4 restorations via hDNA correction presumably occur, they are undetectable in these experiments and, therefore, not included in conversion frequency calculations. Conversion event frequencies at a given site are routinely expressed as percentages throughout the text and in Tables 6-8, 10 and 11.

M. S. WILLIAMSON, J. C. GAME AND S. FOGEL

% Basic conversion (BC) = BC frequency (BCF) X 100

total aberrant segregations total tetrads

x 100 - -

where aberrant segregations = (6:2 + 2:6 + 5 : 3 + 3:5) + 2(ab4:4 + ab6:2 + ab2:6 + ab7:l + abl :7) + 3(ab5:3 + ab3:.i), etc. The first numeral in a segregation ratio refers to the wild-type allele; the second to the mutant allele. Events of the ab6:2, ab2:6, ab7:I or abl:7 type are counted twice, since these segregations are considered to result from two independent events. Ab4:4 segregations are also counted as two events because they occur at a frequency considerably lower than expected if they resulted from single, symmetrically initiated events (FOGEL et al. 1979).

1 5 : 3 I or PMS frequency and the I6:2 I segregation frequency, where I 6:2 I = (6:2 + 2:6) and I 5:3 I = ( 5 : 3 + 3:5). The expressions % I 5:s I and % 1 6:2 I are used to denote these respective coni- ponent frequencies expressed as percentages. The numerator for % I 5:3 I includes all genetically sectored spore colonies, and the numerator for % I 6:2 I includes 6:2 and 2:6 segregations and any segregation in which they are contained (e.g. , ab7:l). PMS represents an event that must be derived from hDNA; the recombination intermediate that gives rise to 1 6:2 1 type segregations need not be exclusively hDNA; it could contain a double-strand gap. The PMS ratio is the fraction of aberrant segregations that involve PMS. It is useful for comparing the fraction of aberrant events with PMS at different sites with variable BCF.

Map distance in centimorgans was estimated using the approximation of PERKINS (1949) for closely linked markers:

The BCF can be usefully partitioned into two component frequencies: the

T + 6(NPD) 2(PD + NPD + T)

Map distance = x 100

PD, NPD and T represent parental ditype, nonparental ditype and tetratype segregations, respectively. Centroniere-linked markers were monitored relative to t r p l , which is assumed to segregate at the first division. Gene to centromere map distances were calculated as (tetratypes per total segregations) X 100/2 (MORTIMER and HAWTHORNE 1966).

RESULTS

Mutant screen Strain design: MW 104-1 B (Figure 1) carries heteroalleles on chromosome ZIZ

for detecting changes in prototroph frequencies in mutagenized survivors. When meiosis is interrupted by plating cells onto vegetative growth medium, the quantitatively determined maximal meiotic prototroph frequencies per plated cell for this strain are 8.2 X lo-', 1.3 X for the his4, thy4 and leu2 allele pairs, respectively. These frequencies reflect the frequencies of both reciprocal recombination and a subset of nonreciprocal recombination events (NICOLAS 1979). It is generally assumed that heteroalleles yielding low prototroph frequencies are physically close, especially if i t can be determined that each member of a pair is a high conversion allele. Jikewise, high prototroph frequencies imply physically distant heteroalleles, especially when these alleles exhibit low conversion frequencies individually. When the segregation of individual alleles is followed in meiotic tetrads, the his4 alleles

and 2.1 X


each exhibit the highest BCF and the leu2 alleles the lowest. Because the reverse relationship is observed for prototroph frequencies, it is reasonable to conclude that the leu2 alleles are widely separated and the thy4 alleles are separated by an intermediate distance relative to the his4 alleles.

Identijication of putative mutants: Among 6000 colonies derived from EMS- mutagenized cells, 27 presumptive mutants displaying elevated meiotic prototroph frequencies at one or more of the three test loci were identified. Often the most significant meiotic prototroph increase was seen at his4. Among these, 12 also displayed elevated spontaneous mutation rates to CAN resistance. Six isolates were chosen for more detailed analysis. Five were mutators, with high and low meiotic prototroph frequency increases for his4 and thr4 alleles, respectively, whereas the leu2 prototroph frequency was not increased. Digenic or monogenic segregations for the mutator phenotype in tetrads from all possible pairwise crosses of haploid derivatives of the five mutator isolates established that four loci were represented. Each of the single mutants express different pleiotropic phenotypes with respect to spontaneous mutation and meiotic gene conversion profiles. The loci identified were previously reported under the gene symbol COR (FOGEL, MORTIMER and LUSNAK 1981, 1983). Here, they are designated PMS, a notation that more appropriately describes a phenotypic aspect rather than an assumed function in meiotic mismatch correction. Experiments involving derivatives of two mutant isolates with alterations in the same gene ( P M S I ) are described below. Effects of pms mutations at three other loci and double mutant interactions will be described elsewhere.

Genetic analysis of pms 1 - 1 and pms 1-2 The segregation pattern for the mutator phenotype with respect to CAN

resistance is illustrated in Figure 2. Tetrad data in Table 3 demonstrate that pmsl heterozygotes exhibit 2:2 segregations for the mutant mitotic mutator phenotype in 88 of 91 tetrads. The three exceptional tetrads probably represent examples of phenotypic overlap (see MATERIALS AND METHODS) or gene conversions. Thus, the mutator phenotype probably reflects a single mutational difference.

Cosegregation of the mutator phenotype and enhanced meiotic prototroph frequency phenotype occurred in 33 of 35 and 22 of 24 dissected spore colonies from pmsl-1 and pmsl-2 diploids tetrasomic for chromosome III and heteroallelic at his4. Since the frequency distributions of the meiotic and mitotic phenotypes in mutant and wild type overlap by 5% yielding occasional misclassifications, the cosegregation data are consistent with both phenotypes resulting from a single mutation. However, these observations do not exclude the possibility that the two phenotypes reflect mutations in two closely linked genes.

Allelism ofpsml-1 and pmsl-2: Table 3 displays tetrad data concerning the mitotic spore colony phenotype of segregants from pmsl-1 X pmsl-2 double heterozygotes. Segregation patterns indicate that pmsl-1 and pmsl-2 are allelic and confer the same mutator phenotype. Whether these two mutations are

618 M. S . WILLIAMSON, J. C. GAME AND S . FOGEL

Y

5 Po

FIGURE 2.-Scgrcgiitioii of the i1iiit;itor ptic.iiocyl)c in t c . t i - i i i I ~ 1.1-oiii ii p m s l - / /P.\ / .Yl f.’:\.\/*/ C A N / ’ diploid. ~l’lie left ;ind riglir Iialvcs of fiicli pliite tlispliiy ;iscosp)ral colo~iy prints froni six dissected asci. (Six spores did not survive.) Prints froni iiscosporal colonies carrying the pms l - I mutation develop CAN-resistant papillae. These reflect an elevated niutation rate ;it C A N I . UV treatment produces a siniilar effect, independent of the PMSI genotype. T w o replicas displaying spontaneous CAN resistance are included to illustrate the reproducibility of the mutator phenotype segrrgiit ion.

TABLE 3

Segregation of the pnisl mutator phenotype in tetrads

!k=gregatioii rlaM

Sr r.i in Genotype 4:o J:1 ‘12 1:s 0:4 T l ~ l ~ l l

MW2101 PMS I /PMS I 33 I 0 0 0 34 MW2IO2 PMS l / p m s I - I 0 0 52 2 n 54 M W 2 I 0 3 PMS I l p m s l - 2 0 0 36 1 n 37 MW2109 pms I - I / pms I - I 0 0 0 0 3 5 35 MW2108 pms l -P /pms l -P 0 0 0 I 19 20 MW2111 , 2112 p m s l - I l p m s l - 2 0 0 1 I 39 41

Scored by estiniating tlie freqiiency of CAN-resistant outgrowths on spore colony prints. All diploids were C A N I / C A N I . Data are given as tetrad ratios (rather than octad ratios) because sectors for the nititator phenotype were not diagnosed. The first nuniber in the tetrad ratio indicatrs the nuniber of spore colony prints with two or fewer outgrowths (wild-type phenotype). and tlie second nuniber indicates the nuniber of spore colony prints with three or niore outgrowths (nititant phenotype).

identical cannot be determined; rare spore colonies classified as “wild type” were found, but these also occur among segregants froni pmsl -1 and pmsl-2 homozygotes. The alleles pms l -1 and p m s l - 2 were isolated from a single mu-


tagenesis treatment without cell division before plating and on this basis are considered independent mutations. However, one or both may have existed prior to EMS mutagenesis.

Spore viability effects: Spore viability is decreased in all pmsl-1 and pmsl-2 homozygotes. For example, among strains used to study pmsl effects on meiotic gene conversion, spore viability is 92% in wild type and 76% of dissected asci yield four viable spores (1 206 asci sampled). Heterozygotes for pmsl-1 or pmsl-2 exhibit spore viability comparable to wild type. In contrast, pmsl-1 homozygotes display 62% spore viability, and only 27% of asci yield four surviving spores ( 1 850 asci sampled). Likewise, 2 1 % of asci have four survivors in pmsl-2 homozygotes, and in a pmsl- l lpmsl-2 diploid used to study pmsl segregation patterns, 27% of asci had four survivors. These observations indicate that the mutant effect on spore viability is recessive.

When pmsl homozygotes or heteroallelic diploids are compared with wild type, the distributions of ascal viability classes are obviously nonhomogeneous (P 5 0.05, d.f. = 4) because asci with four survivors make a smaller contribution to total dissected asci in mutant strains. The distribution of asci with three or fewer survivors is also nonhomogeneous when pmsl homozygotes and wild type are compared (P I 0.05, d.f. = 3). The three-survivor class is un- derrepresented in the mutant, whereas the two-survivor class is overrepre- sented. Haploid-lethal events generated before and during meiosis may both contribute to, and are consistent with, the mutant spore viability pattern. A qualitative decrease in spore viability was observed when zygotic cultures were repeatedly subcultured prior to sporulation.

Map position of the PMSl gene: The PMSl locus exhibits second division segregation in approximately 68% (21 of 31) of the tetrads analyzed relative to trpl-1 and 58% (32 of 55) relative to ura3-1. Thus, PMSl is not centromere linked (MORTIMER and HAWTHORNE 1966). The PMSl gene was mapped to chromosome XZV by the pmsl-1 mitotic mutator phenotype using the spo l l - mapping method and strains developed by KLAPHOLZ and ESPOSITO (1 982a). To establish a more precise map location for P M S l , tetrads were analyzed from strains heterozygous and in repulsion for pmsl-1 and met4, a locus previously located on chromosome XlV by KLAPHOLZ and ESPOSITO (1982b). The data, 33PD:ONPD:6T, demonstrate linkage, and the map distance between PMSl and MET4 is estimated at 7.7 cM. The gene order met4-top2-pmsl was established in a separate three-point cross, and the following map distances were estimated from a sample of 43 tetrads: met4-top2, 8.1 cM; top2-pmsl, 4.7 cM; met4-pms1, 12.8 cM.

Quantitative experiments with pms 1 Mutation rate estimates: Forward mutation rates to CAN resistance at the

CAN1 locus and reversion rates for his4-4 were estimated in pmsl and PMSl strains. Reversion rates of his4-4 and leu2-1 include suppressor mutations as well as intragenic mutations. The pmsl mutator phenotype is most apparent when forward mutation at CAN1 is monitored; nearly a 50-fold increase in mutation rate is observed in pmsl haploids (Table 4). At his4-4, the reversion


TABLE 4

Spontaneous mitotzc mutation rates“

Forward Keversion and/oi mutation to suppression of

Sirain Genotype CAN resistance his4-4

MW2012-6A PMSZ 6.77 X 1.51 X lo-’ MW2012-3B P M S l 6.50 X I O v 8 MW2003-10B pmsl-1 3.06 X 5.62 X IO-** MW2013-4A pmsl -2 3.08 X 4.35 X

MW2013-1D pmsl-2 3.06 X ~ ~~ ~

Rates expressed as events per cell division. * Significantly different from control rate ( P < 0 05)

increase is much lower (four- to five-fold) but statistically significant (P 5 0.05) for pmsl-1 strains.

Rates of mitotic prototroph formation: The spontaneous mitotic prototroph rate at his4 in heteroallelic pmsl diploids increases nearly five-fold, from 8.3 X lO-’/cell division in wild type to 4.1 X 10-6/cell division in the mutant. The rate increase observed in the mutant at thr4 is less than three-fold (3.6 us. 1.6 x 10-’/cell division in wild type). At his#, prototrophs result from heteroallelic gene conversion as well as reversion at h id -4 . The haploid-equiv- alent reversion rate at h i s44 is 2.9 X 10-’/cell division for wild type and 6.3 x lO-’/cell division for pmsl-1. When these contributions are eliminated, the mitotic prototroph rate resulting from gene conversion at his4 is 6.5-fold higher in the mutant (3.5 X 10-6/cell division us. 5.4 X lO-’/cell division in wild type).

Radiation effects in pmsl-1 and PMSl strains: The pmsl mutants are not sensitive to UV or ionizing radiation by patch tests on plates. UV survival curves, shown in Figure 3 , for wild-type and pmsl-1 strains are similar in experiments with either log phase or stationary phase cells. A synergistic effect with respect to UV killing that occurs when certain other mutations affecting meiotic recombination are combined with UV excision-defective mutations (see GAME 1983 for review) was not found for pmsl-1 radl-1 double mutants (data not shown). These observations indicate that PMSl does not function in pyrim- idine dimer repair. In addition, UV-induced mitotic prototrophic recombinant frequencies for thr4 and leu2 allele pairs are similar regardless of the PMSl genotype, but the frequency at his4 is five- to ten-fold greater than wild type at all U V doses in pmsl diploids (Figure 4). This suggests that UV-induced and spontaneous prototrophic recombinant frequencies increase in the mutant when heteroalleles are in close proximity, as at h i d .

Frequencies of meiotic prototroph formation: In MATaliMATa haploids disomic for chromosome IZZ, pmsl mutant prototroph frequencies at his4 and thr4 exceed wild-type values early in the course of exposure to sporulation conditions and remain elevated. This is determined by plating these strains back to selective vegetative media after exposure to sporulation media for various time periods. For example, when cells sporulated for 60 hr resume mitotic growth,


100

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> > U 3 v)

I- 1.0 z w 0 U w a

a -

0.1

0.0 1 1 I I I I I 34 68 136 204 2 7 2 340

uv DOSE (JIM*) FIGURE 3.-UV survival curves for wild-type (e) and p m s l - l (0) diploids. Experiments with

stationary phase cultures (solid lines) and log phase cultures (broken lines) are depicted. Each survival curve represents the average of two or more experiments, usually with different, but closely related, strains.

prototrophic recombinant frequencies in pmsl disomic haploids are identical with controls for the leu2 allele pair but on average are 16-fold higher at his4 and %fold higher at thr4. At this time point, viability is 24% in both mutant and control strains. Frequencies per plating unit (including inviable cells) for pmsl-1 are 1.1 X 4.8 X and 2.1 X for h i d , t h y 4 and leu2 heteroalleles, respectively. The pmsl phenotype is probably not caused by a

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uv DOSE ( J I M ~ ) FIGURE 4.-Effect of UV dose on mitotic prototroph frequency per survivor in pmsl-1 (M) and

wild-type (0) diploids heteroallelic at (a) his4, (bj leu2 and ( c ) thr4. Frequencies reflect those for prototrophic recombinants; values were corrected to eliminate the contribution of prototrophs from reversion.

delay in the transition from commitment to recombination to commitment to the meiotic reduction division in the mutant. If the opportunity for recombination were prolonged and the haploid-lethal meiotic divisions delayed in the mutant, cell survival would be increased relative to wild type until very late in the time course of sporulation. However, survival is the same in the mutant and wild type for all time points in the course of sporulation.

Meiotic prototroph frequencies for diploids are presented in Table 5. The two types of experiments shown represent different protocols and are not directly comparable. Prototroph frequency increases exhibit similar patterns in diploids and disomic haploids but are more pronounced in diploids. On average, prototroph frequencies in diploids homoallelic or heteroallelic for pmsl are 40-fold and 16-fold above controls at his4 and thr4, respectively, after 96 hr of sporulation on plates. Complementation was not observed for pmsl-1 and pmsl-2, a result consistent with their allelism. However, the enhanced meiotic prototroph phenotype is not completely recessive (see Table 5 ) .

The effects of pmsl on meiotic segregations

Data describing the distribution and frequency of aberrant meiotic segregations at 13 singly heterozygous sites from wild type, pmsl heterozygotes and


TABLE 5

Meiotic prototroph frequencies

Strain Genotype His+/survivor Thr+/survivor

623

Plate sporulation”

MW2101 PMSIIPMSI 4.18 X 1.40 X MW2102 PMSI/pmsl-I 1.94 X 2.56 X MW2103 PMSl /pms l -S 7.97 X Revertant MW2109 p m s l - l / p m s l - l 7.18 X lo-’ 1.65 X lo-’ MW2108 pmsl-2/pmsl-P 2.00 X lo-’ 1.69 X lo-’ MW2111 p m s l - l / p m s l - 2 2.20 X lo-’ 4.15 X lo-’ MW2112 p m s l - l / p m s l - 2 2.08 X IO-’ 1.97 X IO-’

Liquid sporulation‘

MW2101 PMSI/PMSI 5.95 X 6.29 X MW2109 pms l - l l pms l -1 1.45 X lo-’ 3.41 X lo-’ MW2108 pms l -2 /pms l -2 1.39 X lo-’ 5.65 X lo-’

a Rates reflect prototrophs per viable spore after 96 hr of sporulation. Premeiotic prototroph background was not determined.

* Unusually high frequency may reflect high premeiotic background.

‘ Rates reflect prototrophs per ascus with one or more viable spores and prototrophs per viable unsporulated diploid cell after 72 hr of sporulation. Rates from these experiments cannot be directly compared with rates obtained after 96 hr of sporulation on plates.

TABLE 6

Aberrant segregations in unselected tetrads from PMSl/PMSl strains ~

Heterorygous Total Total PMS marker 6:2 2:6 5:3 3:5 Other events tetrads %16:21 %15:31 %BC ratio Disparity

~~ ~

MAT trpl-1 ura3-I MAL2 CUP1 leu2-27 thrl-1 thrl

ade8-18 arg4-16 met13 his2

l y s l - 1

4 3 5 4 3 3

25 16 38 17 27 49 63

5 0 0 0 9 7 0 0 0 10 7 0 1 0 13 7 0 0 0 11

1 6 0 0 0 19 7 0 0 0 10 8 2 4 0 39

2 2 2 2 0 42 3 1 0 2 0 71 10 22 18 1“ 69 9 1 2 1 0 58

3 9 0 1 0 89 52 0 0 0 115

874 1.0 0 1.0 0 0.8 0.4 907 1.1 0

908 1.3 0.1 1.4 0.08 0.6 605 1.8 0 1.8 0 0.6 833 2.3 0 2.3 0 0.2 256 3.9 0 3.9 0 0.4 613 5.4 1.0 6.4 0.15 2.3* 629 6.1 0.6 6.7 0.10 0.8 904 7.7 0.2 7.9 0.03 1.2 736 3.7 5.7 9.4 0.61 0.8 634 5.7 3.5 9.2 0.38 0.9 894 9.9 0.1 10.0 0.01 1.2 892 12.9 0 12.9 0 1.2

1.1 0

Homogeneous samples from three wild-type strains (MW154, 193 and 202) are represented. A portion of these data are presented in FOCEL, MORTIMER and LUSNAK (198 1, 1983).

One ab4:4 segregation, scored as two events. * Indicates significant departure from expected ratio of 1.0 (P < 0.05).

pmsl-1 homozygotes are presented in Tables 6-8. To assess frequency changes in meiotic gene conversion within a region, segregation data from two-point crosses at his4 (Figure 6 and Table 10) and three-point crosses at arg4 (Figure


TABLE 7

Aberrant segregations in unselected tetrads from PMS 1 /pms 1 heterozygotes

Hetero7ygour Total Total PMS niarker 6:2 2:6 5:3 3:5 Other events tetrads %16:21 %15:3) %BC ratio Disparity

MAT t r p l - I ura3-I MAL2 CUP1 leu2-27 thr4-1 thrl

ade8- 18 arg4-16 met13 his2

lysl-1

0 4 0 0 0 4 433 0.9 0 0.9 0 NA 0 0 0 0 0 0 509 0 0 0 0 NA

10 0 0 0 0 10 509 2.0 0 2.0 0 NA 1 2 0 0 0 3 349 0.9 0 0.9 0 0.5 1 2 0 0 0 3 419 0.7 0 0.7 0 0.5 2 1 0 0 0 3 289 1.0 0 2.0 0 2.0 9 4 0 0 0 13 347 3.8 0 3.8 0 2.3

13 8 0 3 0 24 419 5.0 0.7 5.7 0.13 1.2 17 19 0 2 0 38 432 8.3 0.5 8.8 0.05 0.8 13 2 11 11 0 37 474 3.2 4.6 7.8 0.60 1.9 4 4 1 2 0 11 130 6.2 2.3 8.5 0.27 0.8

25 20 0 0 0 45 388 11.6 0 11.6 0 1.3 44 27 0 0 0 71 474 15.0 0 15.0 0 1.6*

Combined data sets from six pmsl-2 heterozygotes (MW166, 170, 185, 186, 206 and 205) and

* Indicates significant departure from expected ratio of 1.0 (P < 0.05). two pmsl-2 heterozygotes (MW167 and 314) are represented. NA = not applicable.

7 and Table 11) were compared from mutant and control diploids. These latter studies allowed analysis of gene conversion along a defined chromosomal segment rather than at a single site. Intergenic and gene to centromere intervals were also monitored (Table 9).

Data for wild-type strains in Table 6 illustrate that the %BC at the i 3 heterozygous sites under study is highly variable, ranging from less than 1% to greater than 10%. PMS is infrequent at most sites (observed in less than 0.5% of segregations per site, on average). However, PMS is relatively frequent (3-6%) among segregations for the two markers ade8-18 and arg4-16. Data from pmsl heterozygotes (Table 7 ) indicate that the mutant gene is recessive with respect to its effect on PMS frequencies. Wild-type and pmsl heterozygote data sets represent a statistically homogeneous sample. Taken collectively, the sample includes more than 1000 control segregations for site by site compar- isons with data from pmsl-I homozygotes.

In pmsl-1 homozygotes (Table 8 and Figure 5 ) the divergence from control meiotic segregation patterns is conspicuous for eight of the heterozygous sites, all characterized by low PMS ( I 5:3 1 ) frequencies in wild type. These include t rp l - I , ura3-1, leu-2-27, thr4-I, thrl , l y s l - I , met13 and his2. At these sites, PMS frequencies (expressed as % I 5:3 I ) in the mutant were consistently higher than, and statistically different (P 5 0.05) from, control values at all sites except thr4-1 and t rp l - I . Similar data for pmsl-2 are limited but reflect the same trend and also indicate that leu2-1 is similarly affected. The average %BC for the eight markers listed above is slightly higher in pmsl-1 diploids (7.9 us. 6.1 % for controls). BCF increases are noted at all sites except thr4-1 and metl3. The % I 6:2 I is lower in the mutant than in the controls at all eight sites (3.6 us. 4.7% on average). Significant differences between mutant and control I 6:2 I frequencies are found at three sites, but in general the mutant frequency


TABLE 8

Aberrant segregations in unselected tetrads from pmsl-l/pmsl-1 strains

Heterozygous Total Total PMS marker 6:2 2:6 5:3 3:5 Other events tetrads %16:2) %15:31 %BC ratio Disparity

MAT 4 0 0 0 0 4 524 0.8 0 0.8 0 NA trpl-1 1 1 2 0 0 4 476 0.4 0.4 0.8 0.50 3.0 ura3-1 2 1 4 4 0 11 277 1.1 2.9* 4.0* 0.73* 1.2 MAL2 4 2 0 0 0 6 468 1.3 0 1.3 0 2.0 CUP1 2 2 0 0 0 4 513 0.8 0 0.8 0 1 .o leu2-27 3 3 3 3 0 12 335 1.8 1.8* 3.6 0.50* 1.0 thr4-1 3 1 3 3 0 10 329 1.2* 1.8" 3.0 0.60* 1.5 thrl 16 8 15 6 0 45 464 5.2 4.5" 9.7* 0.47* 2.2* lysl-1 4 1 13 7 0 25 216 2.3* 9.3* 11.6 0.80* 2.1 ade8-18 8 13 13 12 0 46 411 5.1 6.1 11.2 0.54 0.8 arg4-16 2 5 5 2 1" 16 140 5.0 6.4 11.4 0.56 1.3 met13 11 10 2 3 0 26 277 7.6 1.8* 9.4 0.19* 1.0 his2 25 18 26 17 6' 98 474 9.3* 11.4* 20.7* 0.55* 1.5*

Combined data sets from five related pmsl-1 homozygotes (MW170, 200, 201, 347 and 348) are represented. A portion of these data are presented in FOGEL, MORTIMER and LUSNAK (1981, 1983). NA = not applicable.

Segregations scored as two events at arg4-16, one ab4:4; at his2, three ab6:2, two ab4:4 and one abl:7.

* Indicates significant difference between mutant and control values (P < 0.05), except for disparity values where the comparison is to an expected ratio of 1.0.

decreases are small relative to the absolute I 6:2 I frequency in controls. In summary, for these eight highly affected sites, conspicuous I 5:3 1 frequency increases occur, and these are usually accompanied by decreases in I 6 :2 I frequencies that do not completely compensate for 1 5:3 I frequency increases. The values of any or all of the three parameters %BC, % I 6:2 I and % 1 5:3 I may be altered.

Segregation class distributions (derived from data in Tables 6-8), which describe the contributions of 6:2, 2:6, 5:3 and 3:5 events to total aberrant events, are nonhomogeneous for ten of the 13 heterozygous sites studied (P P 0.05, d.f. = 3) in p m s l - 1 mutants compared to controls. Individual conversion class frequencies (based on total segregations) at these ten heterozygous sites are illustrated in Figure 5 to allow comparison of data from p m s l homozygotes and control strains. Significantly different frequencies can be demonstrated for individual conversion classes in the mutant. The frequency changes observed in the mutant generally result in a decrease in the ratio of 6:2/5:3 and 2:6/3:5 segregations relative to controls. In addition, disparity values, calculated as (6:2 + 5:3)/(2:6 + 3:5) (LEBLON 1972a; NICOLAS 1979), are low but show significant departure from 1.0 at thrl (2.1) and l y s l -1 (2.2) in p m s l - 1 diploids but not in controls. Conversely, a significant departure from parity at ade8-18 (1.5) and thr4-1 (2.3) noted for controls is absent in the p m s l - 1 data. These results illustrate that (6:2 + 5:3) vs. (2:6 + 3:5) proportions can also change at some sites in the mutant.

Statistically significant departures from control conversion class frequencies include increases for the 2:6 frequency at ade8-18 and the 5:3 frequency at


trp 7- 7 I '1 I L

6:2 2:6 5:3 3:s

2 1 1

thrrl- 1

ura3- 7 I 2i

4 1 6:2 2:6 5:3 3:5

thr 1

6:2 2:6 5:3 3:5

leu2-2 7 2

6:2 2:6 5:3 3:5

6:2 2:6 5:3 3:5

l aded- 18 8rg.4- 16 4

2

1

6:2 2:6 5:3 3:5 6:2 2:6 5:3 3:5

a

met 73 6 - - E - - - c - e

4

2 1.1 6:2 2:6 5:3 3:5 1

6 : 2 2:6 5:3

his2

/I 3:5

FIGURE 5.-Coinparison of aberrant segregation class frequencies at ten heterozygous sites in p m s l - 1 homo7ygotes and control diploids. Mutant data from Table 8 and combined heterozygote and wild-type control data from Tables 6 and 7 are illustrated. For each set of bar graphs, the x axis displays aberrant segregation classes and the y axis indicates the frequency (among total segregations) X 100. Solid black and horizontally striped bars, respectively, represent mutant and wild-type frequencies for any aberrant segregation class. Vertically striped bars represent mutant frequencies that are significantly different from control frequencies (P 5 0.05).


arg4-16 (Figure 5) in pmsl -1 homozygotes. These mutant shifts are unusual compared to the pattern observed for the eight “highly affected” low PMS markers described above. In wild type, these atypical markers display high %BC (10%) and PMS occurs in approximately 60 and 35% of aberrant events at ade8-18 and arg4-16, respectively (see Table 6 and FOGEL et al. 1979). The arg4-16 mutation is a base substitution G + C transversion, whereas the non- revertible ade8-18 allele is a 38-base deletion (WHITE, LUSNAK and FOGEL 1985). In pmsl -1 diploids, a modest BCF increase at ade8-18 is not accompanied by a PMS frequency increase. The effect is entirely attributable to the 2:6 conversion class frequency increase. At arg4-16 the BCF is slightly elevated and the PMS frequency increases, equaling that observed for ade8-18, with frequency changes occurring in all conversion classes in pmsl -1 diploids. The decrease in 6:2 frequency at arg4-16, accompanied by a larger increase in 5:3 frequency, is similar to the pattern observed at other affected heterozygous sites. In contrast, 2:6 frequency increases accompanied by 3:5 frequency decreases are unique to this site. These results and those from the his4 heteroallelic conversion study (see below) indicate that 6:2 or 2:6 conversion frequencies can either increase or decrease in the absence of PMSl function in a site-dependent manner.

In the same pmsl-1 diploids, gene conversion parameters were unchanged at the remaining sites heterozygous for CUPl , MAL2 and MAT; these are characterized by low %BC (1-2%) and zero PMS in wild type. These three markers probably do not represent base substitution or single base addition/ deletion mutations. Heterozygosity at CUPl is typically reflected by a ten-fold difference in copy number at the CUPl locus (FOGEL and WELCH 1982; WELCH et al . 1983). With respect to structural organization, MAL2 could resemble the SUC genes (CARLSON and BOTSTEIN 1983), and heterozygosity might actually reflect hemizygosity for the gene(s) regulating maltose fermentation (NEEDLE- MAN and MICHELS 1983). At MAT, a 650- to 750-base pair (bp) segment of internal nonhomology distinguishes the two MAT alleles (HICKS, STRATHERN and KLAR 1979; NASMYTH et al. 1981). One sectored (nonmater and mater) colony occurred in a tetrad from pmsl -2 . The sectoring may have resulted from PMS at the MAT locus, but diploidy of the nonmater sector, a result necessary for this conclusion, was not determined.

Intergenic and gene-centromere recombination: Map distances, recorded in Table 9, were estimated from exchange frequencies in tetrads in which both markers that define an interval exhibited Mendelian segregations. Several intergenic intervals on chromosomes 111 and VZIZ and gene to centromere intervals in wild-type diploids and pmsl -1 homozygotes are compared. Map distances are essentially unchanged in pmsl -1 homozygotes relative to wild type. Map distance estimates were comparable in pmsl-1 heterozygotes. These results indicate that pmsl-1 does not affect the reciprocal recombination frequency in the monitored intervals. Because the observed conversion frequency increases are not accompanied by increases in map distance in the mutant, gene conversion and reciprocal recombination appear to be uncoupled. Explanations for these observations are considered in the DISCUSSION.


TABLE 9

A comparison of genetic map distances in pmsl-1 and wild-type difloids

PMS 1IPMSl a pmsl - l /pmsl - lb

Map distance

Map Sample distance Sample

Interval size P NPD T (cM)’ size P NPD T (cM)’

CENVIII-ARG4 432 -325- 107 12.4 445 -307- 138 15.5 ARG4-THRI 314 225 1 88 15.0 428 293 0 135 15.8 T H R l -CUP1 325 131 7 187 35.2 431 172 2 257 31.2

Total VI11 62.6 62.5

HIS4-LEU2 291 199 3 89 18.4 207 142 1 64 16.9 LEU2-CENIII 310 -285- 25 4.0 224 -211- 13 2.9

MAT-THR4 309 181 3 125 23.1 224 137 4 83 23.9 THR4-MAL2 311 85 18 208 50.8 220 60 14 146 52.3

CEN I II-MAT 461 -208- 253 27.4 466 -238- 228 24.5

Totdl 111 123.7 120.5

CENV-URA3 464 -407- 57 6.1 325 -262- 63 9.7

Total 192.4 192.7

“Combined data from MW154, 193 and 202. bCombined data from MW170, 200, 201, 347 and 348. ‘ Calculated according to PERKINS (1949) or from tetratype frequency for centromere-linked

markers.

Studies with his4 and arg4 heteroallelic strains

To characterize the pmsl mutant effect more fully and possibly delineate the role of PMS1 in hypothetical mechanisms for processing recombination intermediates, conversion events were monitored at heteroallelic his4 and arg4 sites. The control data used for statistical comparison represent a homogeneous sample obtained by combining the observations from wild type and pmsl heterozygotes.

Two-point crosses at his4: The alleles his#-4 and his4-519 have not been separated by deletion mapping (FINK and STYLES 1974; CULBERTSON et aE. 1977). Based on physical evidence, the two sites are less than 300 bp apart (G. FINK, personal communication). Data representing 129 1 control and 505 pmsl segregations are summarized in Figure 6 and Table 10. When his#-# (an ochre mutation) is singly heterozygous, 6.3% of segregations are aberrant and 10% of these exhibit PMS (FOGEL et al. 1979). PMS was not observed in more than 200 control segregations with the singly heterozygous frameshift mutation his#- 519 (%BC = 8.6%). In the combined control data from his4 two-point crosses, %BC is 7.4% at either his4-4 or his#-519. Coconversion events spanning both sites (of the co-6:2,2:6 or co-2:6,6:2 type) constitute 98% of the events. This observation implies that the recombination intermediate subtends a region of interaction that usually includes both sites (FOGEL and MORTIMER 1969; FOGEL

629

CONTROL 4 5 19

1291 asci 96 events 505 asci 105 events

FIGURE &-Meiotic conversion events in two-point crosses at h i d . Control data were obtained from three related wild-type strains (814 tetrads) and six strains heterozygous for either pmsl-1 or pmsl-2 (477 tetrads). Mutant data are from five pmsl-1 homozygotes (466 tetrads) and two pmsl-2 homozygotes (39 tetrads). Solid lines represent 16:2 I type segregations and dashed lines represent I5:31 type segregations. Reciprocal crossovers are denoted by "X". The values above each symbol (line) correspond to the number of events seen for each class. The numbers in parentheses show the distribution of these classes; the first number is the number of 6:2 or 5:3 segregations, and the second number is the number of 2 6 or 3:5 segregations. For events involving two alleles, the numbers in parentheses always refer to the segregation of his4-4. Double-event segregations (e.g., ab4:4) are included by classifying their component events separately.

TABLE 10

Summary of conversion events for his4 two-point crosses ~

Conversion class frequency (X 100) PMS

Site Strain 6:2(A) 2:6(B) 5:3(C) 3:5(D) %BC ratio Disparity

hid-4 Control 4.3 3.1 0 0 7.4 0 1.4 pmsllpmsl 4.2 2.8 5.7* 6.3* 19.0* 0.64* 1.1

hid-519 Control 3.1 4.2 0 0.1 7.4 0.01 0.7 pmsllpmsl 3.0 5.5 5.7* 4.6* 18.8* 0.55* 0.9

(C) -t (D) (A) + (B) Total" Control 7.4

pmsl lpmsl 9.7 0.1 7.4 0.01 NA

13.5* 20.8* 0.65* NA

Based on data in Figure 6. A portion of these data are presented in FOGEL, MORTIMER and LUSNAK (1 98 1, 1983). NA = not applicable.

"Column (A) + (B): frequency of events with 6:2 or 2:6 at one or more sites. Column (C) + (D): frequency of events with 5:3 or 3:5 at one or more sites. The BCF is less than the sum of these two frequencies because the same event may simultaneously contribute to values in both columns.

* Significant difference between mutant and control values (P < 0.05).


et al. 1979). The absence of PMS at his4-4 in the two-point control cross is statistically significant compared with the PMS frequency when this site is singly heterozygous, Thus, the presence of the adjacent heterozygosity at hid-519 can suppress PMS at his4-4.

In pmsl homozygotes (pmsl-I and pmsl-2 data combined) the %BC increases approximately 2.5-fold to 19.8% at his4-4 and 18.8% at his4-519 (Table lo). In addition, Figure 6 illustrates that the net %BC increase over controls (+13.4%) within the region defined by these sites is broadly distributed among several possible classes of conversion events, creating a dramatically different distribution of events in the mutant. Single-site events amount to almost 25% of the excess events with excess single-site I 6:2 I and single-site I 5:3 I (PMS) occurring equally frequently at both sites. In contrast, double-site events with I 6:2 I at his4-519 and I 5:3 I (PMS) at his4-4 occur at a significantly higher frequency than double-site events with I 5:3 I (PMS) at his4-519 and I 6:2 I at his4-4 (P 5 0.05). Double-site co-PMS events make up two-thirds of all excess events in the mutants. Finally, the percentage of events involving 1 6:2 I at one or both sites is higher in the mutant than in wild type (9.7 us. 7.4%). Also, the single-site I 6:2 1 event frequency at his4-519, like that at ade8-18, exceeds the control value.

Three-point crosses at arg4: The three arg4 alleles span the 1.4-kb nucleotide- coding region (BEACHAM et al. 1984) of the gene; arg4-3 is proximal. If we assume linearity between meiotic exchange frequency and distance between alleles, arg4-3, located at the low conversion end of the gene, could be separated from arg4-16 by more than I100 nucleotides, with arg4-16 and arg4-36 separated by less than 300 nucleotides at the high conversion end of the gene (based on data from FOGEL, MORTIMER and LUSNAK 1981). The physical distance between his44 and his4-519 may, therefore, be similar to that between arg4-I6 and arg4-36, although wild-type heteroallelic conversion profiles for the two allele pairs are strikingly different.

The %BC for the region defined by the arg4 alleles is 9.3% in control diploids in which 536 tetrads were analyzed. PMS events are relatively rare at all three sites (Table 11). In this three-point cross, the PMS frequency at arg4-16 is significantly lower (% I5:3 I is 1.5 us. 3.3%, P 5 0.05) than when this marker is singly heterozygous. In control strains double-site events involving the alleles arg4-16 and arg4-36 and triple-site events are about equally frequent (Figure 7), i . e . , 20 us. 16. Together they make up 72% of total aberrant segregations. Fourteen single-site events constitute the remaining aberrant segregations and, of these, eight occur at arg4-16. Double-site events involving the arg4-3 and arg4-16 interval are not observed. The BCF at arg4- 3 is about 2.3-fold lower than at the other two sites. Among observed events involving arg4-3, 16 of 18 or 89% include both arg4-16 and arg4-36. Taken together these observations are construed as evidence for a fixed site of recombination initiation at arg4 (FOGEL et al. 1979; FOGEL, MORTIMER and Lus- NAK 1981), possibly localized between arg4-I6 and arg4-36 or distal to arg4- 36.

Compared to controls, conversion events in pmsl-1 diploids are more nu-


TABLE 11

Summary of conversion events for arg4 three-point crosses

63 1

Conversion class frequency (X 100)

Site Strain 6:2(A) 2:6(B) 5:3(C) 3:5(D) %BC PMS ratio Disparity

arg4-3 Control pmsl-1 lpmsl-1

arg4-16 Control pms l - l l pms l -1

arg4-36 Control pmsl-1 lpms l - I

Total" Control pmsl-1 lpmsl-1

0.9 2.1 0.9 1.2

4.3 2.4 1.8* 2.7

2.8 4.5 2.1 2.4

(A) + (B) 8.2 7.8

0 0.4 1.5* 3.6*

0 .4 1.1 4.5* 2.1

0 0.2 2.5* 4.2*

(C) + (D) 1.7

11.8*

~ ~~

3.4 0.11 0.4* 7.2* 0.70* 0.5*

8.2 0.18 1.3 11.1 0.60* 1.3

7.5 0.03 0.6 11.5* 0.61* 0.7

9.3 0.18 NA 18.1* 0.65* NA

Based on data in Figure 7. N A = not applicable. a See footnote a , Table 10. * Significant difference between mutant and control values (P < 0.05). For disparity values, the

comparison is to the expected ratio of 1.0.

merous and varied. Figure 7 illustrates that seven of 13 event types not represented in control data are represented in the pmsl -1 sample, These include coevents at arg4-3 and arg4-16. In the mutant, PMS frequently occurs in double- and triple-site events, whereas comparable patterns are rare in controls. PMS is observed in more than 50% of aberrant segregations at each site. The %BC (1 8.1 %) is nearly double the control value. An excess of single-site events (+7.6%) occurs mainly as PMS single-site events at arg4-3 or arg4-36 and as single-site I 6:2 I events at arg4-16. The +5.6% excess triple- and double-site event frequency, contributed by events with PMS at one or more sites, nearly equals the decreased frequency of coevents without PMS (-4.5%). The co- PMS event frequency at arg4-16 and arg4-36 (5.1%) represents about half the events that include both sites in the mutant. Such events occur infrequently (less than 0.2%) in wild type.

Although the I 6:2 I frequency decreases at individual arg4 sites in pms l diploids, the frequency of events with I 6:2 I at one or more sites is only slightly lower than for controls (7.8 us. 8.2%). In addition, the polarity observed in wild type is diminished in pms l -1 diploids. In the mutant multiple- site conversion events that include arg4-? increase in frequency, and the single- site event frequency at this site increases substantially as well. The %BC for arg4-3 more than doubles, whereas %BC increases for the other two sites are about 1.5-fold (Table 11).

DISCUSSION

Meiotic prototroph frequency estimates in pmsl strains reveal that genetic distances are increased more than 20-fold between adjacent heteroallelic sites,


+------------ARGd

- _ _ - - - - 0 - - - - - - - - - - - - - - 0 - - - - - - - 7(3,4) _ - - - - - - - - - ~ _ _ _ _ - - - - - - - It0 1) . . . . . . . . . . . . . . . . . . . . . .

536 asci 50 events 332 asci 60 events

FIGURE 7.-Conversion events in three-point crosses at arg4. Control data were obtained from one wild-type (247 tetrads) and five pms l -1 heterozygotes (289 tetrads). Mutant data are from four pms l -1 homozygotes. All strains are closely related; most were contructed from segregants of a p m s l - 1 heterozygote. Symbols are described in the legend to Figure 6. For events involving two or three alleles, the numbers in parentheses always refer to the segregation of the left-most affected allele.

whereas the distances between widely spaced alleles remain unchanged. The general effect of the p m s l mutation on meiotic gene conversion is evidenced by increased PMS frequency as well as increased BCF at many heterozygous and heteroallelic sites. At affected individual sites, a decreased I6:2( segregation frequency is typically observed and this decrease is generally smaller than the corresponding PMS frequency increase. However, at some sites the 16:2( frequency is unchanged or possibly increased.

Findings concerning the pms mutants, described here in detail for p m s l , represent compelling genetic evidence for the presence of hDNA meiotic recombination intermediates in yeast. The recent work of DAVIDOW and BYERS (1984) with pacl also demonstrates that hDNA can form during meiosis, at least under conditions of pachytene arrest. Yet, the prevalence of PMS in these mutants does not necessarily indicate that wild-type gene conversion precursors are principally characterized by hDNA. For example a mutational block could allow non-hDNA precursors to be shunted into a minor recombination pathway characterized by hDNA recombination intermediates. An analogous example of a mutational switch is provided by the E. coli recF recombination pathway (CLARK 1973, 1974; ROTHMAN, KATO and CLARK 1975).


Segregation data from pmsl homozygotes suggest that the magnitude of the mutant PMS frequency increase may be correlated with the type of heterozygosity present at individual heterozygous sites. PMSl is clearly required to maintain the low PMS frequencies normally observed at sites heterozygous for certain simple mutations. PMS frequencies are substantially elevated at sites heterozygous for several base substitution mutations (arg4-36", his4-4", leu2-I", lysl-I", trpl-1") and a +1 frameshift mutation, hid-519. At many sites at least 50% or more of aberrant events are PMS. Similar PMS effects observed at six other tested sites heterozygous for "low PMS' markers (arg4-3, his2, leu2-27, met l3 , t h r l , thr4-1 and ura3-1) may indicate that these mutant alleles also represent similar base substitution or frameshift mutations.

More data are required to evaluate the more subtle role of PMSl in processing recombination intermediates at sites heterozygous for "high PMS" markers such as ade8-18 and arg4-16. However, several observations on mutant PMS effects at sites heterozygous for these alleles are noteworthy. The ade8- 18 mutation is a 38-base deletion and arg4-16 is a G - C transversional base substitution mutation (WHITE, LUSNAK and FOGEL 1985). In the pmsl mutant, PMS is observed in approximately 60% of aberrant segregations at either site. For ade8-18, this represents no increase in PMS frequency compared to wild type, indicating that PMSl is not required for processing recombination intermediates at this site. In contrast, it is evident that PMSl plays at least a minor role in the gene conversion mechanism at arg4-16 because the mutant PMS frequency increases two- to three-fold when this site is singly heterozygous or present in triple point crosses.

Conversion profiles are not obviously affected at MAT, MAL2 and C U P l . These heterozygosites involve large nonhomologies, including insertions, deletions or substitutions of at least several hundred base pairs. Because conversion is normally quite infrequent at these sites (%BC < 2%), mutant effects may simply go undetected. Alternatively, recombination intermediates containing large unpaired regions may be processed independently of PMSl function. This might be accomplished by local DNA replication or degradation of single- strand loops formed by strand transfer (RADDING 1979; LICHTEN and FOX 1983). T o further distinguish the PMSl role in the conversion of deletions, studies are planned utilizing large (300- to 500-base) deletions in the his4 gene. In wild type, these deletions convert at a frequency approximately comparable to point mutations in the same region (FINK and STYLES 1974), but PMS does not occur (FOGEL, MORTIMER and LUSNAK 1981).

Marker-specific pmsl effects on PMS are comparable to results from studies of prokaryotic hDNA mismatch correction in Streptococcus pneumoniae. In this context, the pmsl meiotic gene conversion phenotype is most simply inter- preted in terms of an alteration in meiotic hDNA mismatch correction, although alternative interpretations cannot be ruled out (see below). In S. pneumoniae the transformation efficiency of sequences containing different base substitution mutations varies and is controlled by the Hex+ gene (CLAVERYS et al. 1983). In Hex- strains all sequences transform with very high efficiency because mismatches are not removed from hDNA (CLAVERYS, ROGER and


SICARD 1980). Mismatches resulting from transversion mutations identical with arg4-16 normally transform with very high efficiency in Hex+ strains, but transformation efficiency is even higher in Hex- strains. Transformation of deletions is not under Hex+ control. Deletions similar in size to add-18 transform with high efficiency, whereas large deletions transform with low efficiency, regardless of the Hex genotype. By strict analogy the PMSl gene product would be expected to control hDNA misniatch correction mainly at sites heterozygous for certain base substitution and frameshift mutations by a selective excision- resynthesis mechanism.

Any interpretation of the pmsl meiotic phenotype is contingent upon the theoretical framework chosen to describe the mechanism(s) of meiotic gene conversion in wild-type yeast strains. Possible mutational effects predicted by each of two current molecular meiotic recombination models will be considered. Each model posits a distinct and separate mechanism to account for the bulk of observed I6:2 I segregations. In principle the mechanisms described by both models may be operative in the same cell and, thus, contribute to aberrant segregations. However, it is heuristically more useful to consider the pmsl effect on the basis of each model separately.

A recent version of the Meselson-Radding model (MESELSON and RADDING 1975; RADDING 1982), as it applies to yeast, postulates that a recombination intermediate can form, for example, by asymmetric strand transfer to a chromatid containing a single-strand gap. The resulting region of interaction con- sists exclusively of hDNA and is considered to be confined to a single chromatid. On this model hDNA correction is postulated to account for all 16:21 type segregations in yeast. hDNA correction is accomplished by single-strand excision and resynthesis following mismatch recognition. In theory, I6:2 I segregations and cryptic 4:4 restorations both result from mismatch correction events, whereas I5:3 I segregations reveal the absence of hDNA correction. The rarity of PMS in wild-type yeast is considered to reflect an efficient mechanism for hDNA correction.

The DSBR model (SZOSTAK et al. 1983) is based on experiments designed to assess plasmid-chromosome interactions in mitotic cells (ORR-WEAVER, SZOS- TAK and ROTHSTEIN 1981). On this model, recombination initiates after a double-strand gap, flanked by single-strand tails, forms on one chromatid. Strand transfer initiating from the gapped chromatid potentiates strand transfer from the donor chromatid across the the double-strand gap. The major recombination intermediate includes a single-strand gap flanked by hDNA. Unidirectional gene conversion to (6:21 occurs via DNA synthesis on a donor strand template when a mutant site falls within the gap. The DSBR model assumes that mismatch-stimulated hDNA correction occurs as outlined for the Meselson-Radding model, with the important qualification that it is inefficient at sites heterozygous for base substitution mutations in yeast and other ascomycetes. The model accounts for the observation that PMS occurs infrequently in yeast (FOGEL et al. 1979) by postulating that gaps are very long relative to hDNA tails, with 16:2 I gene conversions occurring primarily by double-strand


gap repair. (However, hDNA tails are postulated to be long relative to double- strand gaps in Ascobolus and certain other ascomycetes.)

The pmsf effects on meiotic gene conversion can be rationalized using either model to identify specific mutational alterations that could generate excess hDNA during meiotic recombination. Some alterations will be described below, Explanations derived from the Meselson-Radding model are based on the no- tion that apparent hDNA correction efficiency is decreased as a direct or indirect effect of the pmsf mutation. These explanations are not consistent with the DSBR model which assumes that hDNA correction is ordinarily very inefficient in wild-type yeast. However, the DSBR model can explain the pmsf effect by postulating that a single mutational change creates a longer recombination intermediate with a shorter double-strand gap.

According to the simplest explanation based on the Meselson-Radding model, the pmsf mutants would be deficient in a function required for mismatch correction in meiotic hDNA. The P M S f locus might code for a component of the mismatch correction apparatus or a gene product required for the expression or assembly of the mismatch correction apparatus. Conse- quently, correction tracts initiating near mismatches in hDNA would be shorter and less frequent in the mutant, and excess 15:31 segregations would indicate the relative change in correction efficiency at a particular site. This rationale depends on the critical assumption that silent 4:4 restorations are a reqular, frequent product of mismatch processing in wild-type hDNA. In the mutant, such events would be unmasked and appear primarily in the form of aberrant segregations manifesting PMS. Thus, the mutant BCF increase at a given site represents the net frequency of 4:4 restoration contributed by P M S f mediated correction. 4:4 restoration has been experimentally confirmed in Ascobolus (HASTINGS, KALOGEROPOULOS and ROSSIGNOL 1980). In yeast, its frequency relative to the 16:21 frequency may be a site-specific property (SAVAGE and HASTINGS 198 1 ; HASTINGS 1984).

An alternative explanation based on the Meselson-Radding model accounts for the p m s f phenotype by postulating that heteroduplex recombination intermediates are longer in the mutant. This alteration has two consequences. First, the BCF will increase on average, simply because hDNA is more prevalent during meiosis. In addition, the presence of excess hDNA could account for the reduced 16:21 frequencies found at many sites in the mutant if its presence resulted in an apparent decrease in mismatch correction efficiency among observed conversion events.

A specific p m s f defect based on the DSBR model could involve a delay in the transition from a single-strand gapped precursor to a double-strand gapped precursor, allowing the single-strand gap to become longer than usual and the double-strand gap to be shorter than usual (F. STAHL, personal communication). If, for example, a single-strand specific endonuclease required for double-strand breaks were defective, recombination intermediates would have smaller double-strand gaps on average and the entire region of interaction would be longer than in wild type. Under these conditions fewer 16:21 conversions and more 15:31 conversions would occur at any site on average.


T h e results from heteroallelic conversion studies at his4 and arg4 are consistent with each of the mutational alterations cited above. For example, increased event frequency and changes in the proportions of observed conversion events can be easily accommodated if correction tracts are shorter and less frequent in the pmsl mutant. Events that would normally become silent cores- torations are now observed as single-site events or events with PMS at one or niore sites. Single-site I6:2 I segregations could be generated by two nonoverlapping short correction tracts on strands of opposite polarity (FOGEL et al. 1979; KALOGEROUPOULOS and ROSSIGNOI, 1980).

T h e same heteroallelic conversion effects would be produced if the pmsl mutant had longer recombination intermediates initiating from different fixed sites, with length ranges that overlapped substantially compared to wild type. T h e observed mutant BCF increases at his4 (nearly three-fold) and arg4 (two- fold), and the mutant pattern of single-site us. multisite events are predicted under these conditions. One or more sites would be included in the recombination intermediate more often than in wild type. I f initiatioxl of gene conversion were via double-strand breaks (DSBR hypothesis) event patterns in the mutant are explained by invoking shorter double-strand gaps and longer hDNA tails, the latter being the substrate for mismatch correction, which is inefficient in both mutant and wild type. However, these patterns are suffi- ciently accounted for without invoking the additional complexity imposed by an altered DSBR mechanism. For example, if single-strand gaps (Meselson- Radding model) were the precursors for recombination initiation, then longer hDNA recombination intermediates (excess hDNA) could upset the balance and pattern of hDNA correction reducing apparent correction efficiency.

O n a broader level, the frequency of gene conversion initiation might also be increased in the mutant if we interpret the mutant BCF increase at face value. This alteration could be directly or indirectly related to the mechanistic alteration that decreases the frequency of 16:21 type events while increasing the (5:3) type event frequency. Observed mutant BCF increases are not accompanied by intergenic exchange frequency increases. To explain the mutant BCF increase by an increase in conversion initiation requires the additional postulate that some other factor (unaffected in the mutant) regulates the probability of exchange, i.e., conversion initiation may simply be a precondition for exchange. However, the elevated BCF in the mutant is readily explained without this additional postulate. For example, if the same number of conversion initiations occur in mutant and wild type, but normally silent initiations are unmasked in the mutant because apparent mismatch correction efficiency is reduced, then the mutant conversion event frequency will increase with no effect on reciprocal recombination. To distinguish these hypotheses, further experiments are required to assess the frequency of exchange among conversion tetrads in three-point crosses that define short genetic intervals (<5 cM) between central and outside markers.

T h e PMS phenotype of the pmsl mutants warrants comparison with the gene conversion profiles observed in wild-type Ascobolus, where sites hetero- iygous for base substitution mutations normally display high PMS. In Asco-


bolus the frequency of ab4:4 segregations for such markers is dependent on their location within a gene (PAQUETE and ROSSIGNOL 1978; ROSSIGNOL and HAEDENS 1980). This observation is consistent with recombination initiation by asymmetric strand transfer from a fixed origin, followed by symmetric transfer before termination. In wild-type yeast there is no evidence for symmetrical hDNA based on limited data for “high PMS” markers such as ade8- 18 (FOGEL et al. 1979). Symmetrical hDNA might well contribute to the recombination intermediate in a mutant with altered gene conversion. However, ab4:4 segregations are rare in the pmsl mutant, even at those sites where PMS is frequent. In pmsl-1, two ab4:4 segregations were observed among 3400 segregations at ten monitored heterozygous sites. If recombination intermediates in pmsl strains consisted exclusively of symmetrical hDNA (HOLLIDAY 1964), then more than 20 ab4:4 segregations are expected for this sample. [The expected ab4:4 frequency based on symmetrical hDNA recombination intermediates = a(1 - p)* (FOGEL et al. 1979), where a = the probability of recombination initiation and p = the probability of hDNA correction. To calculate a and p , random correction is assumed, i . e . , I6:2 I type segregations = (4:4 restorations).] The observed ab4:4 segregations can be accounted for without postulating that symmetrical hDNA contributes to the recombination intermediate. In this sample only 3.3 events are expected if ab4:4 result solely from independent superimposed 5:3 and 3:5 events involving asymmetric DNA. [The expected ab4:4 frequency at any heterozygous site can be approx- imated as (0.75) (frequency 5:3) (frequency 3:5). This estimate assumes no conversional interference and that the same chromatid and strand can be involved in both events.] These observations suggest that symmetrical hDNA does not contribute significantly to the recombination intermediate in the pmsl mutant, and they further imply that symmetrical strand transfer does not normally occur in wild-type yeast. The observations are entirely consistent with each of the mutant defects outlined above based on the Meselson-Radding or DSBR models.

PMS frequencies at affected sites indicate that hDNA is generated and remains unprocessed in the pmsl mutant at a frequency characteristic of sites heterozygous for base substitution mutations in wild-type Ascobolus (ROSSIG- NOL, PAQUETTE and NICOLAS 1979). However, the behavior of the frameshift allele, hzs4-519, is inconsistent with Ascobolus conversion patterns. In Asco- bolus, presumptive frameshift mutations typically display very low PMS and extreme (6:2 + 5:3)/(2:6 -t 3:5) disparity (LEBLON 1972a; NICOLAS 1979; ROS- SIGNOL and PAQUETTE 1979). The pmsl mutant conversion profile for his4- 519 is virtually identical with that for base substitution mutations. PMS is observed in about half the aberrant segregations (Figure 6). The disparity ratio at this site (<2.0, Table 10) is similar in wild type and mutant and not nearly as large as disparity ratios in Ascobolus, which can reach 100.

The Meselson-Radding model does not predict which mismatches (e .g . , frameshift, deletion, base substitution) will give rise to PMS when mismatch correction is mutationally affected (directly or indirectly). In addition, the model posits different correction mechanisms in yeast and Ascobolus to ac-


count for the strikingly different wild-type conversion profiles found in these organisms. T h e observation that both frameshift and base substitution mismatches display PMS in the yeast pmsl mutant is consistent with the Meselson- Radding model. In contrast, the DSBR hypothesis presumes that all variation between wild-type conversion profiles in yeast and Ascobolus can be accounted for by differences in hDNA tail length in the recombination intermediate. This economical hypothesis postulates that a yeast mutation like pmsl lengthens hDNA tails (as supposed in Ascobolus) without altering the common mismatch correction mechanism(s) shared by yeast and Ascobolus. It predicts that PMS should be frequent in the mutant only at sites heterozygous for base substitution mutations, whereas PMS should remain infrequent (and disparity should be high) at sites heterozygous for frameshift mutations. This prediction is inconsistent with the observations. In effect, even the DSBR model must explain these observations by acknowledging that wild-type yeast and Ascobolus have different mechanisms for processing mismatches in hDNA recombination intermediates.

Physical and genetic evidence suggest that the hist4-4-his4-519 interval is comparable in length to the arg4-36-arg4-16 interval. Nevertheless, in wild type, the single-site conversion event frequency at the two arg4 sites is much higher than at the his4 sites, suggesting that parameters other than physical distance between sites determine the frequency of single-site events. Some observations concerning the pmsl mutants suggest a possible explanation for this effect. In mutant strains, the BCF increase is larger and the conversion pattern is more highly varied at the his4 sites than at the arg4 sites. For example, single-site events at arg4-I6 and arg4-36 increase less than four-fold from 2.2 to 8.1 ?& of total segregations and at his4 the corresponding increase is 20-fold, from 0.2 to 3.8%. In addition, co-PMS events represent a smaller fraction of excess events at the arg4 sites (39%) compared to the his4 sites (60%). These observations could indicate that the his4 sites are preferred tar- gets for PMSI-mediated coconversion in wild type, whereas other processing functions normally compete more effectively at one or both arg4 sites. In wild type, the genetic distance between the two arg4 sites might be larger because processing frequently includes only a single site.

Although exchange frequencies are normal among surviving spores from pmsl diploids, other observations indicate that this mutation affects chromosomal integrity o r meiotic disjunction. Spore viability is reduced in mutant strains and the patterns of viability in tetrads suggest that lethal events occur at frequencies higher than wild type both before and during meiosis. T h e increased numers of asci with one inviable spore probably signal a meiotic defect in the mutant. T h e reml-1 spore viability phenotype (GOLIN and Espos- ITO 1977) is similar, but this mutation has no effect on meiotic segregation patterns o r meiotic prototroph frequencies.

T h e pmsl mutants are not radiation sensitive. However, they display several pleiotropic mitotic effects. These include somewhat elevated spontaneous and UV-induced prototroph frequencies for close heteroalleles, enhanced spontaneous mutation rates and possibly increased mitotic lethality. That a single


mutation is responsible for the pmsl mitotic and meiotic phenotypes is consistent with the data, although it has not been conclusively demonstrated. The experimental results could reflect a single gene with overlapping mutant and wild-type frequency distributions for the tested mitotic and meiotic phenotypes. Alternatively, each of two closely linked mutations could produce separate effects. If we assume that EMS does not preferentially induce closely linked mutations, the first explanation is more likely.

If the spontaneous mitotic mutator phenotype is attributable to a defect at the same locus conferring the pmsl meiotic phenotypes, then a mutation resulting in reduced mismatch correction in mitotic and meiotic cells could readily account for the mutator phenotype. For example, repair of DNA replication errors and other spontaneous lesions might be inefficient in the mutant (HOL- LIDAY et al. 1979; RADMAN et al. 1979). Thus, in pmsl strains the net effect would be elevated reversion and mutation rates and accumulation of recessive mitotic lethals in diploids. Alternatively, defective double-strand break formation in mitotic cells might produce the same general mutagenic effects if double-strand breaks were required for removal of mutational lesions by recom- binational repair. Either explanation adequately accounts for the smaller reversion rate increase at h i s44 relative to forward mutation rate increase at CANl with the additional constraint that PMSl directs preferred base (or sequence) removal on daughter strands at certain mismatches. The high frequency of mutation at CANl in both mutant and wild type is attributable to the large target size of the gene (WHELAN, GOCKE and MANNEY 1979) estimated to possess the coding capacity for a 280,000-dalton protein. But differences in rate increases at CANl and h i s44 between mutant and wild type are explainable by assuming that misincorporated bases that could result in suppressor mutations or reversions of suppressible mutations are more efficiently removed in the mutant than are the broader spectrum of lesions leading to forward mutation at CANI.

The pms mutations may identify the eukaryotic counterpart to bacterial mismatch correction (repair) systems. Historically, prokaryotic systems have been the focus of investigations concerning the relationship between spontaneous mutation and mismatch correction. The existence of mismatch correction in prokaryotes was established using artificial heteroduplexes (SPATZ and TRAUT- NER 1970; WILDENBERG and MESELSON 1975; WAGNER and MESELSON 1976). In addition, several bacterial mutants appear to be defective in mismatch correction during transformation or recombination. Early studies with S. pneu- monia Hex- (TIRABY and FOX 1973) and E. coli uvrE (mut U ) mutants (NEVERS and SPATZ 1975) indicated that mismatch correction is probably involved in spontaneous mutation because these mutants were also spontaneous mutators. Recent findings with the Hex system (CLAVERYS, ROGER and SICARD 1980; CLAVERYS et al. 1983) were described earlier. The mismatch correction process in E. coli has been genetically dissected with the isolation and characterization of the four mut- mutants (GLICKMAN and RADMAN 1980; RADMAN et al. 1980). These mutants identify a single mismatch correction pathway that acts preferentially on transition and transversion mutations. A higher than normal fre-


quency of wild-type recombinants from a heteroallelic phage lambda cross is observed in mutH strains (GLICKMAN and RADMAN 1980). Similar functions may be identified by pmsl and mutH if hDNA correction tracts are shorter on average in both mutants than in wild type (see WHITE and FOX 1974).

Estimates of the number of loci identified by mutations affecting some aspect of DNA repair, mutagenesis and/or recombination in yeast range from 50 to 100 (for reviews see LEMONTT 1980; HAYNES and KUNZ 1981; GAME 1983). These estimates are tentative at best, since many mutants are pleiotropic and mapping and complementation data are presently incomplete. T h e map position of PMSZ rules out allelism with all mapped yeast genes including the closely linked TOP2 gene, which codes for yeast topoisomerase I1 (DINARDO, VOELKEL and STERNCLANZ 1984), an enzyme required for sister chromatid separation in mitosis. T h e possibility remains that pmsl is allelic to certain unmapped mutator genes identified by mut (HASTINGS, QUAH and VON BOR- STEL 1976; MORRISON and HASTINGS 1979; NASIM and BRYCHY 1979), M C (MALONEY and FOCEL 1980) or rem1 (GOLIN and ESPOSITO 1977) mutations.

T h e genetic characterization of pmsl mutants represents one part of a larger mutational analysis of meiotic gene conversion in yeast. Mutations identified in three other nonallelic PMS loci also elevate PMS, but the meiotic recombination phenotype for each mutant is unique. This work provides a basis for comparative genetic and biochemical studies that will explore the mechanistic details of meiotic recombination in mutant and wild-type strains.

U'e wish to thank DANIEL MALONEY for helpful discussions and critical reading of the manuscript. We also thank M. S. E~POSITO and R. K . MORTIMER for comments on the manuscript. This work was supported by National Institutes of Health grant GM 17317-10 to S. F. M. S. W. was supported in part by a National Science Foundation Graduate Fellowship and National Institutes of Health Training grant GM 07 127. Additional support for J. C. G . was provided by National Institutes of Health grant GM 30990.

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