mitotic recombination in yeast: elements controlling its incidence

Review

Mitotic recombination in yeast: elements controllingits incidence

AndreÂs Aguilera*, SebastiaÂn ChaÂvez and Francisco MalagoÂnDepartamento de GeneÂtica, Facultad de Biologia, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain

*Correspondence to:A. Aguilera, Departamento deGeneÂtica, Facultad de Biologia,Universidad de Sevilla, Avd. ReinaMercedes 6, 41012 Sevilla, Spain.E-mail: [email protected]

Received: 8 February 2000

Abstract

Mitotic recombination is an important mechanism of DNA repair in eukaryotic cells.

Given the redundancy of the eukaryotic genomes and the presence of repeated DNA

sequences, recombination may also be an important source of genomic instability. Here we

review the data, mainly from the budding yeast S. cerevisiae, that may help to understand

the spontaneous origin of mitotic recombination and the different elements that may

control its occurrence. We cover those observations suggesting a putative role of

replication defects and DNA damage, including double-strand breaks, as sources of

mitotic homologous recombination. An important part of the review is devoted to the

experimental evidence suggesting that transcription and chromatin structure are important

factors modulating the incidence of mitotic recombination. This is of great relevance in

order to identify the causes and risk factors of genomic instability in eukaryotes.

Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: Saccharomyces cerevisiae; homologous recombination; hyper-recombination;

double-strand breaks; transcription; chromatin structure; replication defects; DNA

damage; DNA repair

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . 731Mitotic recombination as a source of genetic

instability . . . . . . . . . . . . . . . . . . . . . . . 732DNA damage as a source of mitotic recombination

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733Replication defects as a source of mitotic homo-

logous recombination events . . . . . . . . . . 738DNA damage and replication defects as a source of

recombination in other organisms . . . . . . 739Effect of transcription on mitotic recombination

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739Role of chromatin structure on mitotic recom-

bination . . . . . . . . . . . . . . . . . . . . . . . . 744Concluding remarks . . . . . . . . . . . . . . . . . . 747References . . . . . . . . . . . . . . . . . . . . . . . . 747

Introduction

As a consequence of the importance of homologousrecombination in the generation of genetic variationand its essential role in sexual reproduction, a large

amount of experimental data have emergedthroughout the years regarding conjugationalrecombination in bacteria and meiotic recombina-tion in eukaryotes, particularly fungi. They havemade a great contribution to the understanding ofthe molecular mechanisms of recombination and itsbiological signi®cance.117,122,151,167,179,193,197,200,202

However, beyond its role in sexual reproduction,homologous recombination has a great relevance invegetative growth as a DNA repair mechanism.Mitotic recombination is a ubiquitous process ineukaryotes. It is the basis for gene targeting, forsome human genomic disorders causing geneticdiseases,136 and it has been used regularly as a toolto investigate other biological processes, such as thegenetic basis of development using genetic mosaicsobtained by somatic recombination.64

Although with different biological meaning andgenetic control, both mitotic and meiotic recombi-nation use common factors and steps (reviewed in76, 105, 159, 167, 169, 179, 193, 200, 202). Thegenetic and physical analysis of mitotic and meioticrecombination in S. cerevisiae has provided one

YeastYeast 2000; 16: 731±754.

Copyright # 2000 John Wiley & Sons, Ltd.

important hallmark for all types of homologous

recombination: its initiation by a double-strand

break (DSB). Meiotic recombination is initiated by

a DSB211,212 catalysed by Spo11p.11,101 The DSB is

basically processed by a recombinational repair

mechanism215 that was proposed after performing

experiments of gene targeting in mitotically dividing

yeast cells using gapped plasmids 163. A great deal

of experimental evidence has been provided on the

ability of a DSB to induce a mitotic recombination

event that proceeds via a mechanism that shares all

de®ned hallmarks of homologous recombination:

strand invasion, heteroduplex formation, mismatch

repair, DNA synthesis, and the generation of

Holliday junctions75,105,164,167,193 (Figure 1). Never-

theless, our knowledge on the spontaneous origin of

mitotic recombination is very meagre.Our purpose is to review the data, mainly from

the budding yeast S. cerevisiae, that may help to

understand the spontaneous origin of mitotic

recombination and the different factors that maycontrol its incidence. Most of this review will bebased on results reporting a stimulation of mitoticrecombination (hyper-recombination), some ofwhich were partially reviewed previously.5 A list ofall known S. cerevisiae genes from which, to ourknowledge, mutations with a signi®cant sponta-neous hyper-recombination phenotype have beenreported, is shown in Table 1. We will cover thoseobservations suggesting a putative role of replica-tion defects and DNA damages as sources ofmitotic homologous recombination. However, themain focus of this review will be the experimentalevidence suggesting that transcription and chroma-tin structure are important factors modulating theincidence of mitotic recombination, since there areno recent reviews devoted to this subject. We willcover very few aspects of the mechanisms of eitherhomologous recombination or DSB repair in yeast,as a considerable number of recent reviews areavailable.75,77,159,164,167,197

Mitotic recombination as a source ofgenetic instability

Recombinational repair of a DNA sequence uses anhomologous partner to resynthesize the damagedregion. In a diploid yeast cell, the homologouspartner can be either the homologous chromosomeor the sister chromatid, whereas in haploid yeastcells the homologue should be a sister. As aconsequence, recombination should not lead toany genetic rearrangement. It should have nogenetic consequences if using a sister chromatid or

Figure 1. DSB repair model for recombination. The classicmodel by Szostak et al215. involves the invasion of the 3k-endsinto the homologous DNA molecule to form two hetero-duplex regions and two Holliday junctions. Repair of theheteroduplex determines whether or not a gene conversionevent takes place. Single-strand annealing between the twoinvading and resynthesized DNA strands can re-establish theoriginal molecular con®guration without involvement ofcrossing over.167,216 Resolution of the Holliday junctiondetermines whether or not a crossing-over occurs167,215

Figure 2. Recombination events can occur between DNArepeats located in the same chromatid (a), in homologouschromosomes (b), in non-homologous chromosomes (c) orin sister chromatids (d)

732 A. Aguilera, S. ChaÂvez and F. MalagoÂn

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 731±754.

it should only lead to a mitotic recombination event

between homologous chromosomes, usually a gene

conversion unassociated with crossing-over.As evidenced by the sequencing of the yeast

genome, S. cerevisiae contains a signi®cant amount

of duplicated chromosomal regions and stretches of

repeated DNA sequences that are potential sub-

strates of recombination192,237 (Figure 2). Since

Jackson and Fink95 and Klein and Petes106 reported

that recombination could occur between repeated

copies of a gene arti®cially introduced into one

chromosome, many different types of DNA repeat

constructs have been used to study mitotic recom-

bination in yeast (reviewed in 169). Particularly

interesting was the observation that recombination

could also occur between ectopic copies of one gene

arti®cially introduced into non-homologous chro-

mosomes,96 as was previously reported for the

naturally occurring CYC1 gene S. cerevisiae43 or

between the naturally occurring copies of tRNA

genes in the ®ssion yeast Schizosaccharomyces

pombe.152

Contrary to recombination between homologous

chromosomes and sister chromatids, recombination

between repeated DNA sequences located in either

the same or different chromosomes can have drastic

consequences for the stability of the genome. It can

lead to gross chromosomes rearrangments such as

deletions, inversions or translocations,5,107 even

though many such rearrangements may occur by

aberrant replication or by non-homologous end-

joining (NHEJ).33,79

Among the different types of rearrangements that

can be generated by interchanges between repeated

DNA sequence, deletions occurring between long

interspersed repeats deserve particular attention.

They can arise by recombination events such as

cross-over between two repeats, unequal sister

chromatid exchange or unequal sister chromatid

gene conversion181 (Figure 3), that presumably

follow a standard mechanism of DSB recombina-

tional repair. In addition, deletions could theoreti-

cally also occur by non-conservative events such as

one-ended invasion10,174 or single-strand annealing

(SSA)54,55,129,165 (Figure 4). Sister-chromatid repli-

cation slippage could also lead to deletions.133 It is

likely that these speci®c mechanisms responsible for

deletions between direct repeats can explain the

differential effect of some mutations on deletions

vs. other types of exchanges.

DNA damage as a source of mitoticrecombination

Double-strand breaks

Different experimental evidence suggests that DSBs

act as initiators of mitotic recombination. Thus, it is

well known that DSB-inducing agents such as

X-rays and c-rays induce recombination in

yeast.44,45,84 Since the work by Orr-Weaver et al.,163

indicating that a plasmid linearized with a restric-

tion endonuclease is very ef®ciently integrated into

the chromosome by homologous recombination,

and the ®nding that MAT switching occurred by a

DSB catalysed by the HO endonuclease,205 many

other studies have been performed in the yeast

S. cerevisiae, in which DSBs have been produced

in vitro by restriction endonuclease120,147,162,174

or in vivo by the site-speci®c endonucleases

HO54,55,157,165,182,183,208 and I-SceI173, the Flp

recombinase175 and restriction endonucleases.125 In

Figure 3. Recombination events responsible for deletionsbetween non-tandem direct repeats: intrachromatid cross-ing-over, unequal sister chromatid reciprocal exchange andunequal sister chromatid gene conversion, all of themoccurring presumably by the DSB-repair model shown inFigure 1

Mitotic recombination in yeast: control of incidence 733


Table 1. S. cerevisiae genes for which mutations with a spontaneous hyper-rec phenotype have been described

Gene Function of gene product References

CDC5 Ser/Thr-protein kinase required for exit from mitosis 82

CDC8 Thymidylate kinase 82CDC6 Initiation of DNA replication 25,82

CDC9 DNA ligase 61,177

CDC14 Protein phosphatase involved in cell cycle control 82CDC20 WD-40 protein involved in microtubule function 82

CDC23 Component of anaphase promoting complex, APC 82

CDC45 Initiation of DNA replication 86

CDC73 RNA pol II-associated protein 195CTF4 (CHL15) DNA polymerase a binding protein 115

DNA2 ssDNA-dependent ATPase, DNA helicase and endonuclease 53

DUN1 Protein kinase involved in regulation of DNA repair genes 49

HPR1 Transcription and genomic stability 2,3HSM3 MSH1 homologue 51

MCM1 Transcription factor of the MADS box family 42

MCM2 Component of the MCM complex of initiation of replication 239

MCM3 Component of the MCM complex of initiation of replication 239MCM4 (CDC54) Component of the MCM complex of initiation of replication 86

MCM5 (CDC46) Component of the MCM complex of initiation of replication 86

MCM7 (CDC47) Component of the MCM complex of initiation of replication 86MEC1 DNA damage checkpoint protein 226

MMS21 DNA-repair protein 150,177

MRE11 (RAD58) DSB-repair ssDNA endo- and dsDNA exonuclease 6

PAF1 RNA pol II associated protein 195PAT1 Topoisomerase II-associated protein 230

PKC1 Protein kinase C1 88

PMS1 mutL-like DNA mismatch repair protein 235

POL1 (CDC17) DNA polymerase a 2,82,134POL3 (CDC2) DNA polymerase h large subunit 2,68,82

POL30 PCNA 30

POL31 (HYS2) DNA polymerase h small subunit 210PRI1 DNA primase small subunit 132

PRI2 DNA primase large subunit 132

RAD2 NER ssDNA endonuclease 119

RAD3 DNA helicase of TFIIH involved in NER and transcription 66,138,149RAD5 Snf2p family of DNA helicases 128

RAD6 Ubiquitin conjugating enzyme involved in error-prone repair 103

RAD18 DNA-binding protein involved in DNA repair 14,128,143

RAD9 DNA damage checkpoint protein 48RAD24 DNA damage checkpoint protein 118

RAD27 (RTH1) ssDNA FLAP endonuclease I 201,214,222,226

RAD50 DSB-repair protein 141

RAD51 RecA-like DNA strand exchange protein 1,127,144RAD52 DSB recombinational repair protein 8,140

RAD54 DSB-repair DNA-dependent ATPase 144

RAD55 DSB-repair DNA-binding protein 144RAD57 DSB-repair protein 144

RFA1 ssDNA-binding replication protein 198,199

RFC2 Replication Factor C 160

SGS1 RecQ-like DNA helicase 63,232SIR2 DNA-silencing protein 69

SOH1 Putatively related with RNA pol II transcription 46

SPO11 DSB transesterase of initiation of meiotic recombination 24

SPT4 Chromatin-related protein involved in transcription 139SPT6 Chromatin-related protein involved in transcription 139

SRS2 (HPR5) DNA helicase 2,166

SSL1 Component of TFIIH 138



all cases, reciprocal exchange and gene conversion,as well as deletions, have been detected. This,together with data indicating that meiotic recombi-nation was initiated by DSBs,76,105,179,193,202 has ledto the belief that DSBs are the main event initiatinga recombination reaction that would proceed viathe DSB repair mechanism (Figure 1).

Once end-joining was shown to be an alternativemechanism of DSB repair in mammalian cells,similar evidence was provided for this type ofrepair in yeast, especially after the identi®cation of

the yeast homologues of the mammalian Ku70 andKu80 proteins (reviewed in 33, 50, 99, 126, 224,233). Despite the different relevance of recombina-tion and end-joining in yeast vs. higher eukaryotes,it seems clear that both types of mechanisms areused for DSB repair, the importance of eachpathway being dependent on the stage of the cellcycle in which the DSB is repaired.87,100 Inexponentially growing yeast cells, recombinationseems to be the most prominent DSB-repair path-way, as deduced from the strong sensitivity to DSB-

Figure 4. Non-conservative intramolecular mechanisms of recombination leading to deletions: single-strand annealing andone-ended invasion cross-over

Table 1. Continued

Gene Function of gene product References

SSL2 (RAD25) DNA helicase of TFIIH 119THO2 Transcription and genomic stability 172

TOP1 DNA topoisomerase I 31,124

TOP2 DNA topoisomerase II 31

TOP3 DNA topoisomerase III 9,229TRF4 Required for cell viability in the absence of Topo I 185

XRS2 DSB-repair protein 92



inducing agents of recombinational-repair mutantsvs. end-joining mutants. Indeed, X-ray sensitivity ofKu mutants are only clearly observed in doublemutant backgrounds containing an additionalmutation in a recombinational repair gene, such asrad52.17,18,135,148,194 The question arises as towhether or not the primary event leading to aspontaneous recombination event in mitosis is aDSB or another type of DNA damage or replica-tion error.

In mitosis, evidence that recombination is spon-taneously initiated by DSBs is provided by the factthat all genes required for spontaneous mitoticrecombination (RAD50, RAD51, RAD52, RAD54,RAD55, RAD57, RAD59, XRS2, MRE11 andRFA1) are required for the repair of DSBs inducedby c-ray.84,169

The involvement of DSBs in spontaneous recom-bination may also explain the hyper-recombinationphenotypes conferred by mutations in RAD50,MRE11, XRS2. Since religation of DSBs by end-joining requires these genes, it is likely that moreevents are shunted into the recombinational repairpathway in their absence.6,28,92,223 The possibilitythat hyper-recombination in these mutants isproduced as a consequence of a failure to repairdamage from a sister chromatid has also beenraised.167 In any case, the explanation may not bethat simpleÐotherwise hyper-recombination shouldhave been found associated with mutations inHDF1 and HDF2, encoding the Ku70 and Ku80yeast homologues.

Interestingly, rad51, rad54, rad55, rad57 and rfa1mutants are hyper-recombinant for direct repeatrecombination leading to deletions.1,127,144,198 Sincethe wild-type copies of these genes are required forrecombination, these results suggest that deletionsmust occur by a speci®c type of mechanismindependent of those gene products. This may besingle-strand annealing (SSA). Therefore, DSBsthat are not repaired by a gene conversion/recipro-cal exchange type of homologous recombination(see Figure 1) can be shunted into repair by a strandresection mechanism that does not require aRAD51-dependent strand exchange reaction, pre-sumably single-strand annealing (SSA) (Figure 4).This would explain the speci®c hyper-rec phenotypefor deletions in these rad mutants. Interestingly,RAD59, another gene involved in a RAD51-inde-pendent pathway of DSB repair, is involved in theformation of deletions between direct repeats.93

Mutations in RAD59 confer a slight increasein recombination between homologous chromo-somes.7 This has also been shown for a particularrad52 allele that shares other phenotypes withRAD59-loss-of-function mutations.8 Therefore, itseems that DSBs can also be channelled intodifferent DNA recombination repair pathways,either RAD51- or RAD59-dependent. In addition,recombinagenic breaks might also be channelledinto recombinational repair pathways not leading toa detectable genetic exchange. Thus, to explain thehyper-recombination phenotypes of the checkpointmutations rad9, Fasullo et al.48 have proposed thatthe RAD9 checkpoint may channel the repair ofDSBs into sister-chromatid exchange (SCE), there-fore impeding a detectable mitotic recombinationevent between two homologues. Such an impedi-ment should be released in rad9 mutants.

Single-strand breaks

Whether any type of DNA break can eventuallylead to mitotic recombination without being pre-viously processed into a DSB has been addressed intwo different studies. By using a gIIp-based system,the group of J. Strathern has shown that single-strand DNA breaks also induce recombination.206

However, since the DNA molecule where the breakwas produced acted as receptor of information,their results suggested that the recombinationevents proceeded according to a DSB recombina-tional repair model.215 It is likely that single-strandbreaks were processed into DSBs before recombina-tion took place. Galli and Schiestl59 observed that,whereas gIIp-induced single-strand breaks had noeffect on recombination between direct repeats inthe G1 or G2 stage of the cell cycle, recombinationwas increased when induced during the S phase.Instead, I-SceI-induced DSBs increased recombina-tion in both G1 and G2. Again, these results suggestthat the single-strand break induces recombinationif processed into a DSB, and that processing into aDSB especially occurs when a replication forkarrives at the site of the nick during S phase.

DNA damage other than DNA breaks

Many pieces of evidences suggest that DNAdamage other than DSBs can be channelled intothe recombinational repair pathway. This can bededuced from many hyper-recombination mutantsso far analysed in yeast (Table 1). Thus, sponta-



neous DNA damage susceptible to nucleotideexcision repair (NER) or error-prone repair mayexplain hyper-recombination in a number ofmutants affected in NER and error-prone repair, ifthe unrepaired damage is channelled into recombi-national repair. This can be the case of the hyper-rec phenotypes of the NER mutations in RAD3,149

RAD25119 or SSL1138 or other repair mutationssuch as hpr5/srs22,166,180 rad1814,128,143 or rad6.103

Increased heteroallelic gene conversion caused bydun1 has also been explained by proposing thatDNA-damage-induced gene expression mediated byDUN1 channels the repair of UV damage into anon-recombinagenic repair pathway.49 It is likelythat such damage is processed into a DNA breakbefore being channelled into recombination.

The work of Jinks-Robertson's group213 isparticularly revealing for the evaluation of theimportance of DNA damage other than DSB as asource of mitotic recombination. By inactivatingNER or base excision repair (BER) in yeast cellsby mutations, a strong hyper-rec phenotype isobserved that is synergistic when both BER andNER pathways are simultaneously inactivated. Thehyper-rec phenotype is enhanced if oxidizing agents,such as hydrogen peroxide or menadione, are addedto the cells. In all cases, recombination is dependenton the RAD52 gene. These results suggest that anytype of spontaneous damage may lead to DNAbreaks that need to be repaired by homologousrecombination. It is likely that, in wild-type strains,part of the spontaneous damage produced bymethylation, oxidation and other chemical reactionsescape their main routes of repair, such as BER orNER. The subsequent action of DNA replication,or the partial action of an endonuclease at the siteof the damage, may lead to a subsequent recombi-nagenic DNA break.

The induction of recombination by differentDNA-damaging agents such as UV, alkylatingagents, cysplatin, etc. can be explained in similarterms.20,34,37,41,80 They produce a DNA lesion thatmay be processed into a DNA gap or break. Insome cases, replication may be involved. Thus, UV-induced thymidine dimers or 3-methyladenine, thatcannot be traversed by the replicative DNA poly-merase, can become a DSB during replication.Indeed, the block of the replication fork has beensuggested to be one of the causes of increasedmitotic recombination after 8-methoxypsoralen andUVA irradiation,37 or after cisplatin treatment.80

DSBs are observed after post-treatment incubationand may be responsible for the hyper-recombination observed at the ARG4 locus inmitosis. This is consistent with the observationindicating that interstrand cross-links requireRAD51 to be repaired94 or that the resistance tothe alkylating agent methyl methane sulphonate(MMS) requires all the RAD50 series of genesinvolved in recombinational repair.41,84,169 DSBscould also be generated by the un®nished action ofexcision repair as an intermediate in the reaction.However, since the replication fork is blocked bythe adducts, it is likely that a large proportion ofthose DSBs occur by blockage of the replicativeDNA polymerase (see below). Indeed, the inductionof recombination by UV, MMS, EMS and 4-NQOhas been shown to depend on cell division, suggest-ing (once more) that the recombinagenic event mustbe produced after the replicative polymeraseattempts to go through the unrepaired lesion.60

Mismatches

Mismatches may also stimulate mitotic recombina-tion, as can be deduced from the observation thatplasmid integration is stimulated by mismatches.240

It has been proposed that mismatches couldpotentially lead to recombinagenic structuresduring mismatch repair (MMR) such as single-strand gaps or denatured DNA regions.240 On theother hand, the meiotic studies of Borts andHaber,15 using multiple heterozygosities, suggeststhat a heteroduplex containing multiple mismatchesmay trigger new recombination events. Therefore,it is possible that mismatches can initiate recom-bination events, either directly or after processinginto a DNA break. Nevertheless, we still needmore experimental evidence to con®rm such apossibility.

A different hyper-recombination phenotyperelated to mismatches is that caused by mutationsin PMS1235 and other genes involved in MMR suchas MSH2.30,97,191 The mismatch repair proteinsrecognize mismatches formed in the heteroduplexDNA occurring during recombination and act as abarrier for recombination. In heteroduplexesformed between homeologous sequences, thosebarriers would be very abundant; thus mismatchrepair would play an anti-recombinogenic role. As aconsequence, the inactivation of mismatch-repairgenes would allow heteroduplex to form between



homeologous DNA sequences and recombinationto be completed, explaining the speci®c enhance-ment of recombination between homeologous DNAsequences. This enhancement of recombination canbe explained by extension of on-going recombina-tion events, rather than by a stimulation ofinitiation events.

Replication defects as a source ofmitotic homologous recombinationevents

Replication defects may be one of the main sourcesof mitotic recombination. The accumulation ofeither blocked replication forks or DNA gaps byincomplete replication trigger a post-replicativerecombinational repair pathway that uses a homo-logous partner to bypass the unreplicated region.Thus, it has been shown, in yeast, that the inhibitorof replication hydroxyurea (HU) induces recombi-nation in dividing cells but not in G1- or G2-arrested yeast cells.58 This suggests that the inhibi-tion of replication must lead to recombinageniclesions, presumably DNA gaps or breaks. It is likelythat single-stranded DNA breaks caused as aconsequence of HU treatment frequently lead toDSBs.145,146 The increase in short repeat intrachro-mosomal recombination observed at palindromicstructures may also be the consequence of thedif®culties of the replicative polymerase to progressthrough stem structures.67,85,131,184

Good evidence that replication errors can be animportant source of mitotic recombination comesfrom the analysis of mutants in the DNA replica-tion machinery (Table 1). They show a strongincrease in mitotic recombination between eitherhomologous chromosomes or repeats, whetherintra- or inter-chromosomal. The ®rst replicationmutations shown to lead to strong hyper-recombination phenotypes in yeast were those inthe structural genes of DNA ligase I,61,177 thymidy-late kinase82 or the large subunits of DNApolymerases I2,82,134 and III.2,68,82 Subsequently,hyper-recombination has also been shown formutations in the small subunits of DNA polymer-ase III,210 or the structural genes of the two primasesubunits132 or the replication factors A198 and C.160

Mutations in genes involved in initiation of replica-tion have also been shown to lead to spontaneoushyper-recombination. This is the case of mutations

in MCM2, MCM3, MCM4, MCM5 and

MCM7,86,239 CDC6,25,82 CDC4586 or the DNA

helicase gene DNA2 with a putative role in replica-

tion.53

It is likely that stimulation of recombination in

all replication-defective mutants is provided by the

accumulation of gaps during the S phase of the cell

cycle. Those gaps can become DSBs by subsequent

nicking of the DNA template (Figure 5). This may

also be the case for the mec1 and pol30 alleles that

are lethal in a rad52 background. They accumulate

single-strand intermediates that presumably become

DSBs.145,146 Thus, the 3k-end would be able to

initiate a strand invasion process into a homologous

sequence that would result in a recombination

event. The recent work on mutations of RAD27, a

ssDNA Flap endonuclease, involved in the removal

of Okazaki fragments is very illustrative in this

sense. These mutations show a strong hyper-rec

phenotype178,201,222,226 and are synthetically lethal

with any mutation of the RAD52 series of genes

involved in DSB recombinational repair.214,222

After displacement of the Okazaki fragment by the

DNA polymerase, lack of removal of the 5k ¯ap

structure by Rad27p results in a single-strand gap

susceptible to breakage on the template strand,

resulting in a DSB.222

The recent work on E. coli by the group of B.

Michel showing that the RuvB helicase (involved in

Figure 5. DSB formation as a consequence of replicationfork blockage121. Either a nick occurs in the template of thelagging strand or the two nascent DNA strands re-anneal toform a Holliday junction with a DSB end



the migration of Holliday junctions) is required forthe repair of replication blocks, suggests that Holli-day junctions may be one of the intermediatesformed when replication is impeded.190 The nascentleading and lagging strands can reanneal, by actionof RuvB, to form a Holliday junction and a double-strand end that could initiate a recombination eventthat would require resolution of the Hollidayjunction (Figure 5). Although no evidence of thisprocess has been reported yet in yeast, it isparticularly interesting the accumulation of Holli-day junctions in the rDNA region that are formedin the absence of the strand-invasion proteinRad51p during the S phase of the cell cycle.243

Such Holliday junctions can be explained perfectlyas intermediates of DNA replication. The results ofKadyk and Hartwell,98 indicating that most recom-binational repair occurs between sister chromatids,is consistent with the idea that an important sourceof spontaneous recombination may come fromreplication. It is likely that some of the replicativeerrors are shunted into homologous recombinationevents that use a homologous chromosome as atemplate.

Lack of topoisomerase activity is another impor-tant source of mitotic recombination in yeastthat might be related to replication. Hyper-recombination has been observed in top1, top231

and top3 mutants9,229 as well as trf4,185 pat1230 orsgs1232. Whose gene products have a functional orphysical relationship with Top1, Top2 and Top3,respectively. Consistent with these data, DACA (N-2[-dimethylamino)ethyl]acridine-4-carboxamide), aninhibitor of topoisomerase II, is a recombinagenicdrug to which rad52 cells are very sensitive.52 It ispossible that topoisomerases are involved in DNAreplication termination, as suggested for topoisome-rase III.62 However, it is also likely that the hyper-rec phenotype associated with top mutations isrelated to the role of topoisomerases in otheraspects of DNA metabolism, such as transcriptionor DNA topology by itself (see the section on DNAstructure and topological constraints).

DNA damage and replication defects asa source of recombination in otherorganisms

Studies on the different factors inducing recombina-tion in vegetative cells have been reviewed in

other organisms from bacteria to mammaliancells.26,65,90,137,207,219 Data on mitotic homologoushyper-recombination in eukaryotes other than theyeast S. cerevisiae are very scarce. In the ®ssionyeast Sz. pombe, there are only few hyper-recmutants described.73,74,196,220 Studies on recombina-tion induced by DNA-damage-inducing agents,such as X-rays, or hyper-recombination conferredby mutations in genes involved in DNA repairpathways, other than recombination repair, in E.coli, are abundant. Documentation can be found insome recent reviews.32,197

The role of replication defects in recombinationhas been extensively studied in prokaryotes. The®rst hyper-recombination mutants of E. coli werefound to be affected in different DNA replicationfunctions, including DNA ligase, polymerases orthymidylate kinase, more than 20 yearsago.114,142,242 Indeed, many of the studies per-formed in the yeast S. cerevisiae owe much to theprevious work on E. coli. Information on hyper-recombination in E. coli can be found in severalreviews.12,97,110,111,122,137,197 In general, there is astrong parallelism between hyper-recombination inbacteria and yeast. This parallelism suggests thatyeast studies should become an important referencein order to understand the factors that stimulate theincidence of mitotic recombination and geneticinstability in higher eukaryotes.

Effect of transcription on mitoticrecombination

A connection between mitotic recombination andgene transcription has been well established in yeastas well as prokaryotes and higher eukaryotes.In yeast, both RNA polymerase I- and poly-merase II-driven transcription are known to beinvolved in the induction of mitotic recombinationand genetic rearrangementsÐdeletions, reciprocalexchanges, gene conversions and microsatellitedestabilization.

Mating-type switching is probably a clear exam-ple in which, given the developmental control oftranscription and recombination of the genesinvolved (MAT, HML, HMR and HO), the inter-connection between both processes has to be tightlycontrolled. It has been well established that silen-cing impedes recombination at the HML and HMRloci.77,167 Recently, it has been shown that there is



an in¯uence of transcription on recombination thatis exerted at a distance, independently of thetranscriptional status of the donor and acceptorsequences, by the action of a recombinationalenhancer regulated at the transcriptional level.238

As this aspect of the in¯uence of transcription onrecombination has been recently reviewed,78 we willfocus on other examples, which deal with systemsthat show different recombination levels dependingon their transcriptional state.

RNA polI stimulated recombination: HOT1

The ®rst evidence for transcription-induced recom-bination in yeast was obtained after the isolation ofthe recombination hotspot HOT1102 as a cis-actingelement able to stimulate both intra- and inter-chromosomal recombination. HOT1 corresponds toa 4.6 kb rDNA fragment containing the 3k-end ofthe 25S gene, the 5S gene, the two non-transcribedspacers and the 5k end of the 35S rRNA. Roeder'sgroup,228 using his4 direct repeat intrachromosomalassays, demonstrated that a 570 bp fragment con-taining the initiation site of the 35S rRNAprecursor (I element) and an enhancer of transcrip-tion by RNA polymerase I (E element) were enoughto stimulate recombination. Both I and E elementshave identical behaviour in transcription andrecombination assays. That transcription, promotedby HOT1, is indeed responsible for the stimulationof recombination, has been shown by the facts thatdeletion mutations in the E or I element abolishboth the transcription promotion and recombinatorstimulation204 and that HOT1 activity is lost in aRNA polymerase I mutant incapable of transcrip-tion of the 35S RNA gene.89 It is certainly possiblethat transcription-driven recombination is an indir-ect consequence of the effect of transcription onDNA, caused by the unwinding of the DNA duplexand changes in the local supercoiling or in chroma-tin structure. Interestingly, when Voelkel-Meimanet al.228 inserted an RNA pol I transcriptiontermination between HOT1 and adjacent sequences,the stimulation of recombination was abolished.This indicates that transcription needs to proceedthrough at least one repeat unit to promotestimulation of recombination. This result excludesthe possibility that putative changes in chromatinstructure occurring and/or propagating from thepromoter region are the only factor responsible forrecombination enhancement.

A better knowledge of the biological elementscontained in the HOT1 sequence has provided newclues for the understanding of its recombination-stimulating activity. One of the non-transcribedspacers (NTS1) contains a site called RFB thatblocks the replication fork in the direction oppositeto that of transcription of the 35S RNA gene.21,130

Kobayashi et al.108 has found that the minimalregion essential for the replication block activity liesin the same E element required for transcriptionand recombination enhancement. Interestingly,Huang and Keil89 have shown, by deletion andsite-directed mutation analysis, a good correlationbetween recombination and transcription in theHOT1 region, except for a deletion of a segmentthat is exactly the same as RFB and that greatlyreduces HOT1 activity, with little effect on tran-scription. Kobayashi and Horiuchi109 have identi-®ed FOB1 as a gene encoding a trans-factorrequired for RFB activity. Mutations in this geneabolish the Hot1 phenotype. As they suggest,Fob1p is likely involved in blocking the replicationfork through the RFB site, resulting in enhance-ment of recombination. However, how the RFBactivity is related to transcription to stimulaterecombination remains to be elucidated (see later).Finally, an important feature of the HOT1 sequenceis its ability to greatly stimulate deletions (100-fold).This contrasts with its weak effect on gene conver-sions.228 In any case, the in¯uence of HOT1 onrecombination has been investigated outside of itsrDNA original context.

Stimulation of recombination by RNA polII-mediated transcription

Reports showing that RNA polymerase II-dependent transcription also stimulates recombina-tion appeared after the identi®cation of HOT1.Rothstein's group showed that recombinationevents in directly repeated sequences of the GAL10gene lead to deletions, but not to gene conversions.The frequency of these deletions is increased byRNA pol II-driven transcription.218 As in the caseof HOT1, the level of stimulation of recombinationcorrelates with the level of expression of the repeatsystem. This suggests that recombination may beinitiated in either one of the repeats or theintervening region. The RAD52 gene and, to alesser extent, RAD1 are required, although therecombination pathway that remains in a rad52



rad1 double mutant is positively affected bytranscription as well. It is likely that recombinationis stimulated at the level of initiation and not byimproving the ef®ciency of the recombinationmachinery.217

Another relevant case of RNA pol II transcrip-tion induced recombination is the induction ofTy ectopic recombination, which is mainly non-reciprocal. When transcription of a marked Ty copyis under the control of the GAL1 promoter, its rateof recombination is highly stimulated by transcrip-tional induction. This stimulation is synergistic withthe effect caused by increased levels of donor TycDNA.155 Again, RAD1 and RAD52 are needed forthe transcriptional enhancement of recombination,but mutations in these genes do not completelyabolish the different types of events seen followingtranscription.156

Since transcription driven by both RNA poly-merases I and II can stimulate different forms ofrecombination, an interesting question is whethertranscription at any promoter is able to stimulate acertain type of exchange event. A study by Brattyet al.19 suggests that this may not be the case.Recombination of a chromosomal ade 1 allele witha plasmid-borne ADE1 ORF was stimulated whentranscription of the latter was induced by the GAL1promoter, but not when induced by the promoter-enhancer region of the rDNA locus (RNA pol I) orthe ADH1 promoter (RNA pol II).19 These resultspoint to an in¯uence of the promoter sequence onthe recombination event, at least for plasmid-chromosome exchange.

Recombination associated to RNA polII-transcription elongation: HPR1 and THO2

Although numerous examples exist connectingtranscription and recombination, the nature ofthat connection has not yet been elucidated. Onepossibility is that there is an indirect effect oftranscription on recombination by increasing theaccessibility of DNA to damaging agents or to therecombination machinery. The identi®cation andanalysis of hyper-recombination mutants haveprovided new clues for the understanding of sucha connection. One particularly relevant mutant ishpr1. Originally identi®ed in a genetic screen ofhyper-rec mutants, hpr1 shows a strong increase inthe rate of deletions occurring at direct repeats withno effect on gene conversion or sensitivity to DNA-

damaging agents, as shown for HOT1- and GAL10-stimulated recombination.3,4 The null hpr1 mutantshows pleiotropic phenotypes, such as temperature-sensitivity and reduced levels of transcription ofdifferent reporter constructs,46,241 which suggests aninvolvement of Hpr1p in transcription. Consis-tently, suppressors of the ts phenotype have beenmapped in structural genes related to RNA poly-merase II-dependent transcription.47,225 The isola-tion of mutations in the genes SRB2 and HRS1/MED3 as suppresors of the hyper-recombinationphenotype of hpr1D provided a ®rst link betweenthe phenotypes of transcription and recombina-tion.170,187,188 Both SRB2 and HRS1 encode sub-units of the mediator complex of the RNApolymerase II holoenzyme,113,153,171 and are fullyrequired for the hyper-rec phenotype conferred byhpr1D.187

The transcription-recombination link in hpr1appeared stronger after experiments showing thatthe hyper-rec phenotype of the hpr1 mutants isdetected in direct repeats containing an interveningsequence that is transcribed. Insertion of the CYC1transcription terminator downstream of the DNArepeat that is transcribed, so that transcriptioncannot get through the intervening region, abolishesthe hyper-recombination phenotype.176 Theseresults suggest a role of transcription elongation,rather than initiation, in stimulating recombination.Indeed, a transcriptional analysis of hpr1 mutantsindicates that Hpr1p has a functional role intranscriptional elongation.29 The importance oftranscription elongation impairment on recombina-tion is con®rmed by the fact that the hyper-recphenotype of hpr1 strongly depends on the natureof the transcribed intervening sequence. Recombi-nation levels are extremely high (almost 100%frequencies) when the RNA pol II proceeds throughDNA sequences in which transcription elongation isstrongly impaired (i.e. lacZ). Indeed, there is acomplete correlation between the reluctance of aparticular DNA sequence to be transcribed in hpr1mutants and the ability of such a sequence toinduce recombination when inserted between directrepeats.29

The conclusions reached by studying hpr1 strainshave been corroborated by the identi®cation andcharacterization of mutations in THO2, a geneisolated as a high-copy suppressor of hpr1. As is thecase of hpr1D, tho2D is strongly hyper-rec, it showsan even stronger impairment of transcriptional



elongation than hpr1 and indicates a completecorrelation between impairment of transcriptionand recombination in a given sequence.172 Byconstructing recombination systems under the tran-scriptional control of the GAL1-regulated promoterlocated outside of the repeats, it has also beendemonstrated that the hyper-rec effect of tho2 isonly observed when transcription is active.172

In addition to the stimulation of deletions causedby hpr1 and tho2 mutants, other forms of geneticinstability (such as plasmid loss) have beenobserved in association with the impairment oftranscription elongation. In both types of mutants,the stability of centromeric plasmids is reducedwhen transcription of a plasmid-borne gene isactivated, their levels of instability being dependenton the sensitivity of the transcribed sequence to thehpr1 and tho2 mutation.29,172 These results can beinterpreted as if the hyper-recombination phenotypewere not mainly the consequence of an increase inthe recruitment of the recombination machinerybut, rather, an increase in DNA damage orreplication errors able to initiate recombinationwhen occurring between repeats. If no repeats arepresent, the stability of the replication unit becomescompromised.

Integrated view of transcription-associatedrecombination

Can transcription elongation impairment explainother cases of transcription-induced recombination?Additional studies would be necessary to give ade®nitive answer, but some details suggest that thismight be the case. In all cited cases, transcription isnot marginally present in one locus. Instead, theelongating RNA polymerase traverses either onerepeat unit or the whole recombination system; thisis the case for HOT1.228 In the GAL10-dependentrecombination assay of Thomas and Rothstein,218

the promoter lies between the repeats and istranscribed outwards from the repeat unit towhich it is fused. Interestingly, in all cases inwhich recombination was stimulated, an mRNAwas found that initiated at the GAL10 promoterand entered the bacterial DNA sequences of theintervening region. Although the importance of thistranscript has not been evaluated in connectionwith the stimulation of deletions, it is noteworthythe parallelism between the presence of such atranscript and the need for transcription to occur

through the sequences ¯anked by the repeats inhpr1 and tho2 in all cases in which recombination isstimulated. Another important coincidence ofHOT1, GAL10 and hpr1- and tho2-dependentrecombination is the strong stimulation of dele-tions, whereas little or no effect is observed on geneconversion. We now know that deletions occurringbetween direct repeats are the result of a non-conservative recombination event, presumablysingle-strand annealing or one-ended cross-over.This has been shown for hpr1D mutants186 and itis presumably the situation for the other casesreported. The most important feature that differ-entiates deletions from other recombination events,such as gene conversions or reciprocal exchanges, isthat they can be initiated in the heterologous region¯anked by the repeats.54,55,147,174,208 As observed inhpr1 and tho2D strains, transcription-inducedrecombination is mostly initiated at the region¯anked by the repeats, where transcription elonga-tion impairment is believed to occur. This could bethe case of HOT1- and GAL10-promoted recombi-nation, even though initiation of recombination atthe DNA repeat that is transcribed also occurs.Finally, in all cases, there is a similar dependencyon RAD1, RAD10 and RAD52 of the induceddeletion events.156,186,204,217 Taken together, thesedata suggest that the type of recombinationmechanism triggered by transcription could be thesame in all cases reported.

It has been proposed that stalled or blockedtranscription complexes can trigger recombinationevents.29,176 It might confer nuclease hypersensitivesites as a consequence of an open chromatinstructure or partially unwound DNA, it mightfacilitate the attack of DNA damaging agents, orit may interfere with the replication machinery.More experiments are required to solve this puzzle.However, it is worth discussing the hypothesis thattranscription-associated recombination might beinduced by an arrest of the replication fork aftercolliding with a stalled or blocked transcriptioncomplex29,176 (see Figure 5). Although originallyproposed for hpr1 recombination events,176 thishypothesis could be extended to the other casesreported. As mentioned earlier, HOT1 activityrequires RFB replication fork blocking activity.Indeed, mutations in the FOB1 gene, required forRFB activity, abolish the Hot1 phenotype.38,89 Thiscertainly suggest that HOT1-induced recombinationis mediated by replication fork blocking. Transcrip-



tion elongation may ®rst provoke the collision with

the replication fork and the RFB activity would

then be required for blockage of replication, so that

a deletion would occur by either single-strand

annealing, one-ended invasion or even sister chro-

matid replication slippage.Other types of genetic rearrangements can be

potentiated by transcription in yeast. The most

relevant is the destabilization of simple repeti-

tive DNA sequences. GAL1-induced transcription

through a poly GT tract destabilizes it four- to

nine-fold.234 The effect of transcription is signi®cant

even in msh2 and pms1 mismatch repair-de®cient

mutants, indicating that the observed phenomenon

cannot be explained by a reduction in the ef®ciency

of DNA mismatch repair, but is very likely due to

an increase in the error rate of DNA polymerase. It

would certainly be interesting to know whether

microsatellite instability and deletions of long DNA

repeats are the result of the same molecular event in

connection with transcription. As suggested for

hpr1- and tho2-stimulated recombination events,

the interference of the transcription machinery

with the replication fork during elongation is an

attractive alternative to explain microsatellite

instability.

Transcription-induced recombination in otherorganisms

The interconnection between transcription and

recombination is not exclusive to yeast. On the

contrary, it seems to be a general phenomenon

observed from bacteria to mammals. Some impor-

tant examples are mentioned in this review. The

®rst description of a transcription-induced recombi-

nation process was the Rpo-mediated recombina-

tion of phage lambda in Escherichia coli.91 Rpo

recombination is induced by RNA polymerase, is

recA-independent, and is distinct from Red, Int,

RecBC and RecE pathways. The cross-over occurs

within a narrow region of the phage genome that is

actively transcribed. If the transcribed region is

extended by a rho mutation, affecting transcription

termination, the cross-over region is extended in

parallel, indicating that RNA chain elongation is

the transcription step required for recombination in

bacteria also.91 Other examples of transcription-

facilitated homologous recombination have been

described by studying specialized transduction by

phage l and generalized transduction by phages T1and T4.39,40

Deletions produced by illegitimate recombinationin E. coli are also induced by transcription. pBR332derivatives carrying a pTac promoter suffer dele-tions at a 10x2 rate. Deletion formation is stronglydependent on the strength of the promoter, thetranscript length, and the relative orientation of thepromoter and the origin of replication.227 Indeed,B. Michel's group has suggested that these are theresult of a collision between converging replicationand transcription machineries.227

In the yeast Sz. pombe, transcription driven fromthe ADH1 promoter has also been shown tostimulate recombination,72 consistent with theresults of S. cerevisiae. In mammalian cells, thereare different examples of transcription-inducedrecombination. Thus, it has been shown that atranscribed target site improves the chances of genetargeting in human cells221 or that two integratedcopies of neomycin genes recombine, in Chinesehamster ovary cells, between three- and seven-foldmore frequently when one of the repeats istranscriptionally induced. Interestingly, both directand inverted repeat systems are stimulated and, inthis case, most of the events are gene conver-sions.158

The most relevant case of the in¯uence oftranscription on recombination in animals is prob-ably the assemble of the variable region ofinmunoglobulins and class-switching. The transcrip-tional activity of ¯anking sequences plays a directrole in regulating V(D)J recombination.13 Theelimination of a transcriptional enhancer and apromoter causes a 20- to 100-fold reduction in thefrequency of rearrangements, whereas the elimina-tion of a transcriptional silencer causes a ®ve-foldincrease.123,161 The molecular mechanism by whichtranscription stimulates V(D)J recombination isnot clear, but it has been proposed that transcrip-tional activity increases the accessibility of RAGrecombinases to their substrate sites (RSS). Con-sistent with this hypothesis, a study on the cleavageaccessibility of RSS in different cell types anddevelopmental stages shows that cell-type speci®cchromatin structure determines the targeting ef®-ciency of the RAG proteins and that chromatinstructure plays a role in the allelic exclusionmechanism.203 Class switch recombination has alsobeen shown to be enhanced by transcription.36 Inthis case, RNA: DNA hybrid formation has been



proposed to be an intermediate in the process ofstimulation.35

Therefore, it is clear that the transcriptionactivity and/or the transcription machinery providea scenario in which DNA exchanges are stronglyfacilitated in organisms from bacteria to mammals.However, since neither transcription and recombi-nation in vivo involves naked DNA but, rather, ahigher order protein±DNA structure (chromatin ineukaryotes), we cannot ignore the fact that the ®rstconsequence of transcription is the remodelling ofchromatin structure. Chromatin structure affectsrecombination in both mitosis and meiosis. We willreview these effects in mitosis only. As we will see, itis certainly possible that the effects of chromatinand transcription on mitotic recombination areconsequences of similar types of DNA disturbance.

Role of chromatin structure on mitoticrecombination

As already mentioned, transcription-driven recom-bination may be an indirect consequence of theeffect of transcription on chromatin. Thus, unwind-ing of DNA, or changes in the local supercoilingcaused by transcription, might be responsible forrecombination stimulation. Since recombinationoccurs between DNA molecules organized inchromatin, differences in chromatin structure canexplain the opposite behaviour of two giventranscription units. It is plausible that a change inchromatin structure facilitates the access of recom-bination proteins, leads to hypersensitivity tonucleases and endogenous DNA-damaging agents,or stimulates a pairing reaction. Chromatin struc-ture is therefore an important factor to beconsidered when trying to analyse the effectof transcription on recombination and geneticrearrangement.

Although invoked frequently to explain results,evidence connecting chromatin organization andrecombination is scarce. One good example is HOendonuclease-induced mating-type switching. Haberand collaborators have investigated the require-ments for the different elements of the recombina-tion apparatus in this process of gene conversion.They found that, when the HML or HMR donorsadopt the usual silenced and inaccessible chromatinstructure, the gene conversion event requiredRad51p, Rad52p, Rad54p, Rad55p and Rad57p.

However, when the donor is not silenced andlocated on a plasmid, Rad52p alone is enough tocarry out the recombination event.209 They con-clude that the other Rad proteins, not needed whenthe donor is episomal, are required to facilitatestrand invasion into the otherwise innaccesibledonor sequences. This result re¯ects different needsfor the recombination machinery to complete arecombination event that depends on the chromatinstatus of the DNA.

SIR2

A very good example of the role of chromatinstructure on recombination is the effect of tran-scriptional silencing on the reduction of bothmitotic and meiotic intrachromosomal recombina-tion between the tandem repeats of the rDNA locus.The key protein mediating this repression is Sir2p,which is also involved in maintaining transcrip-tional repression at the silent mating type loci andtelomeres.69A sir2 mutant shows a 10- and 15-foldincrease in recombination that is speci®c for rDNAduplication. This increase in recombination isstrictly dependent on Rad52p (and on Rad50p inmeiosis), whereas the basal recombination in SIR2strains is independent of those functions, suggestingthat Sir2p is involved in excluding the rDNA locusfrom the recombination apparatus.69 Consistentwith this idea, Esposito and collaborators havealso demostrated, by assays of sensitivity to micro-ccocal nuclease and dam methyltransferase, thatSir2p is required for a more closed chromatinstructure in two regions of the rDNA repeats.56

rDNA silencing and chromatin accessibility respondto SIR2 dosage and, at the same time, silencinginversely correlates with recombination, indicatinga double effect of closed chromatin in repressingboth transcription and recombination.56 The differ-ent requirements of basal and sir2-induced recom-bination for RAD52 argue against transcriptionalactivity as an intermediate between chromatinstructure and recombination. In accordance withthis, the sir2 mutation seems not to in¯uence HOT1activity.

SPT/SIN

In the previous cases, chromatin structures seems toin¯uence recombination by conditioning the ef®-ciency of the cellular machinary for DNA transac-tions within the locus. Both cases re¯ect how



silenced DNA (heterochromatin) is a barrier toboth transcription and recombination. A differentstudy using intrachromosomal repeat system sug-gests that chromatin alterations in the euchromatinmight also be responsible for higher levels ofrecombination. MalagoÂn and Aguilera139 measuredthe recombination rates of nine different mutantsaffected in SWI/SNF and SPT/SIN genes. TheSWI/SNF genes code for the subunits of one ofthe main chromatin remodelling complexes neededfor transcriptional induction of a broad set ofgenes,168 whereas SPT/SIN genes encode proteinsthat in¯uence chromatin structure in a negative wayfor transcription.236 The spt4 and spt6 mutationshave a hyper-recombination phenotype that coversreciprocal exchanges, gene conversions and dele-tions between direct repeats. The broad range ofrecombination events suggests that the effect of themutations is exerted at initiation and that ade®ciently packed chromatin can be more accessibleto recombinagenic agents.139 An interesting ques-tion, yet to be answered, is whether the hyper-receffect of spt4 and spt6 is mediated by transcription,as Spt4p and Spt6p have been involved in chroma-tin-mediated repression of transcription.236 SPT6has been shown to contact with histone H3 and tocontrol chromatin structure.16 In addition, Winstonand colaborators have found a direct relationshipbetween SPT4, SPT5 and SPT6 and transcriptionelongation by RNA polymerase II in vivo.83 Adifferent question emerging from these results iswhether the hyper-rec phenotype of the spt muta-tions is also mediated by transcription elongation.In this sense, it is noteworthy that the hyper-recmutation hpr1 also displays genetic interactionswith mutants affected in chromatin structure.Thus, deletion of SIN1, encoding an HMG1-likeprotein, partially suppresses the transcriptionalphenotype of hpr1, and elevated gene dosage ofindividual histones shows severe growth defects incombination with hpr1.241

DNA structure and topological constraints

Another aspect of DNA structure that has beenrelated to mitotic recombination is the topologicalstate of chromatin. The ®rst genetic approach tothis ®eld was made by Fink and collaborators.31

They found that mitotic recombination in therDNA locus, but not in RNA pol II-transcribedloci, is stimulated in the absence of topoisomerase

activities. Both top1 and top2 mutants show 50- to200-fold higher frequencies of recombination rela-tive to wild types. The same results were basicallyobtained by Kim and Wang104 who, in addition,observed that the deleted copies of rDNA in a top1top2 mutant accumulate as extrachromosomal ringsthat can be integrated back into the chromosome iftopoisomerase activity is recovered. Both groupspostulate the accumulation of topological stressresulting from a high transcriptional activity as apossible explanation for the observed hyper-recombination. Topoisomerase activity (either I orII) is, in fact, essential for RNA synthesis,22 as it isrequired for transcriptional elongation by RNApolymerase I.189 This requirement is strengthened ina mutant lacking subunit A34.5 of RNA polymer-ase I, as it becomes quasi-essential in a top1background.57

Mutants affected in TOP1 and/or TOP2 genesare not reported to be hyper-rec in systemstranscribed by RNA polymerase II. This is inagreement with the observation that topoisomerasesI and II are not essential for RNA polymeraseII-dependent transcription.22 However DNA-supercoiling can be accumulated to a certainextent by RNA polymerase II activity in themutant background,23 suggesting that accumulationof DNA supercoiling per se is not suf®cient toinduce recombination. One additional factor thatdifferentiates the effect of topoisomerase mutantson transcription by RNA polymerases I and II isthe behaviour of the chromatin templates. In theabsence of topoisomerases I and II, a severetranscription-dependent chromatin transition canbe detected in rDNA, as analysed by nucleasedigestion and psoralen cross-linking, but not in agene strongly transcribed by RNA polymerase II.27

A disrupted chromatin structure under topologicalstress might therefore be the optimum scenario fortranscription-stimulated recombination. The differ-ent behaviour of RNA polymerases I and II in thisrespect opens the possibility of the existence ofredundant topological activities operating in RNApolymerase II-dependent transcription.

It is certainly signi®cant that the third eukaryotictopoisomerase, Top3p, was uncovered by mutationsconferring hyper-recombination between directrepeats in yeast. Repeated sequences and therDNA region are very instable in top3 mutants aswell. TOP3 codes for a topoisomerase similar tobacterial type I enzyme.229 Mutants affected in



TOP3 show a very poor growth phenotype that canbe suppressed by mutations in a second gene, SGS1,encoding a reverse gyrase activity.63 Sgs1p interactsphysically with Top3p and Top2p, and sgs1 muta-tions also suppress the instability in the rDNA locuscaused by top3. Based on this observation, it hasbeen proposed that Top3p acts on DNA substratesmade by Sgs1p.62 SGS1 is, in fact, homologous totwo human genes involved in genome disordersleading to premature aging: the Bloom's and theWerner's syndrome genes.231,232 Mutations in SGS1also show a broad hyper-rec phenotype, as theypresent an increased frequency of interchromoso-mal homologous recombination, intrachromosomalexcision recombination, and ectopic recombina-tion.63,232 Altogether, these results indicate that thecomplex formed by Top3p and Sgs1p is veryimportant for maintaining genome integrity. HowTop3p and Sgs1p, as well the other topoisomerases,affect recombination is yet to be elucidated. Thework of Harmon et al.81 on the E. coli homologuesTopIII and RecQ suggests that the catenation/decatenation activity of Top3 would be required tosuppress recombination between repeats. TheSgs1p±Top3p complex would suppress recombina-tion by its strand passage activity.63,81 When tworeplication forks collide, the unreplicated interforkregion would become highly underwound andinaccessible to Top3p making the helicase activityof Sgs1p necessary for Top3 to access the regionand decatenate the replicated DNA.

Finally, speci®c DNA regions have been shownto act as mitotic hot-spots for recombination. Thisis the case of the 1.5 kb region found by Neitz andCarbon.154 This is an AT-rich DNA sequence that,when introduced into a linear plasmid, causes theaccumulation of linear forms of the plasmids,suggesting that it may act as a target for DSBs,whether or not mediated by nucleases. It wouldcertainly be interesting to know which particularfeature makes this sequence behave as a hot-spotfor mitotic recombination.

Effect of chromatin and DNA structures inrecombination in other organisms

Not many examples are available about the in¯u-ence of chromatin organization on recombinationin non-yeast systems. Among those we have alreadymentioned, the study on the cleavage accessibility ofRSS in different cell types and developmental stages

in V(D)J recombination shows that cell-type speci®cchromatin structure determines the targeting ef®-ciency of the RAG proteins.203

The possibility that supercoiling can generaterecombinagenic DNA structures has also beenstudied in E. coli. Kohwi and Panchenko112 haveshown that direct repeat sequences separated byeither 200 bp or 1000 bp and containing 25±30 bppoly(dG)-poly(dC) sequences in the 5k region of apTac promoter induce homologous recombinationwhen transcription is activated. In this scenario,negative supercoiling is accumulated at the 5k regionof the promoter, inducing the formation of a triple-helix structure at the poly(dG)-poly(dC) tract. Theypostulate that such a triple structure causesenhancement of recombination, because no hyper-recombination was observed when the dG tract wasnot present. The intramolecular triples would foldDNA, bringing the repeated DNA sequences intoproximity, so facilitating recombination. Althoughthis model may be valid for proximal direct repeatsin bacteria, it is not clear whether it can explain thecases of transcription-induced recombination inyeast, in which direct repeats are separated bymore than 10 kb and are placed in the context ofchromatin.

Finally, although eubacterial chromatin is quitedifferent compared with the eukaryotic DNAorganization, published mechanistic informationabout the effect of nucleosomes on recombinationreactions comes from in vitro experiments withreconstituted nucleosomes and bacterial recom-bination proteins. Thus, Kotani and Kmiec116

have observed that transcription activates RecA-promoted homologous pairing of nucleosomalDNA. On the other hand, Grigoriev and Hsieh70

have shown that a histone octamer blocks branchmigration of a Holliday junction when reconstitutedonto an appropriate DNA molecule. They subse-quently demonstrated that the Holliday junctioncannot migrate through the nucleosomal core unlessDNA±histone interactions are completely dis-rupted. Grigoriev and Hsieh70 propose that, duringrecombination, enzymes are required not only toaccelerate the intrinsic branch migration but also tofacilitate the passage through chromatin. They haverecently shown that the E. coli RuvAB protein isable to drive the migration of a Holliday junctionthrough a nucleosome in the presence of ATP.71

These results predict that eukaryotic cells havedeveloped biochemical functions to allow recombi-



nation to proceed through chromatin. It is certainlyattractive to think that the transcriptional machin-ery may well do such a job.

Concluding remarks

Mitotic recombination is one form of DNA repairthat preferentially uses sister chromatids to repairDNA breaks, many of which may be generatedduring replication or by DNA damage. The analysisof hyper-recombination mutations and the effect ofDNA-damaging agents on recombination is a verygood approach to understanding the molecularbasis of the origin of mitotic recombination.Initiation of recombination in mitosis may beaffected by many elements and processes such asNER, BER, replication, transcription and chroma-tin structure. Particularly intriguing is the connec-tion with transcription and chromatin structure; itis important that these processes be deciphered atthe molecular level to obtain a complete knowledgeof the mechanisms of recombination and theirrelevance in the stability of the genome in yeastand other organisms. The systematic functionalanalysis of the yeast genome provides a novelapproach to identifying new hyper-recombinationmutants. However, we still need much more geneticand molecular analyses to understand both hyper-recombination and the molecular basis of initiationof spontaneous mitotic recombination. This is ofgreat relevance in order to identify the causes andrisk factors of genomic instability in eukaryotes.And yeast are, certainly, the best eukaryotic systemfor studying mitotic recombination in vivo.

Acknowledgements

We would like to thank F. Prado for reading the manuscript

and W. Reven for style correction. This work has been

funded by the EU (BIO4-CT97-2294) and the Ministry of

Education and Culture of Spain (Grants PB96-1350 and

BIO98-1363-CE).

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