the journal of vol. 269, no. 44, issue by printed in u.s.a ... · 27434 sos-independent inducible...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 44, Issue of November 4, pp. 27433-27440,1994 Printed in U.S.A.

UVM, an Ultraviolet-inducible RecA-independent Mutagenic Phenomenon in Escherichia coZi*

(Received for publication, June 28, 1994, and in revised form, August 24, 1994)

Vaseem A. PalejwalaS, Gagan A. PandyaS, Opinder S. Bhanotg, Jerome J. Solomon§, Holly S. Murphy*, Paul M. DunmanS, and M. Zafri HumayunSll From the Wepartment of Microbiology and Molecular Genetics, UMD-New Jersey Medical School, Newark, New Jersey 07103-2714 and the $Department of Environmental Medicine, New York University School of Medicine, A. J. Lanza Research Laboratories, Long Meadow Road, Zhxedo, New York 10987

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or in- accessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior W irradiation of ArecA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVlM (W modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase HI, we have examined mutagenesis at an ZC residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in W-irradiated recA+, and in ArecA cells. These data indicate that W irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap- filling DNA synthesis as well as during normal DNA replication.

A majority of point mutations may arise from misreplication events occurring at sites of DNA damage. Most mutagen-induced DNA lesions are noninstructive in the sense that template instruction is either missing k g . abasic sites) or inacces- sible due to steric reasons (e.g. bulky adducts). Noninstructive lesions lead to replication arrest and are presumed to be lethal unless replication is forced past such sites. Since replication in the absence of accessible template instruction is by definition error-prone, cellular survival is hypothesized to come “at the cost of mutagenesis.* For two decades, the SOS hypothesis (1-4) has been central to our understanding of error-prone DNA synthesis across noninstructive lesions (5-10). According to this hypothesis, replication arrest at noninstructive lesions induces the expression of proteins that act at the stalled replica-

CN-113 and National Institutes of Health Grant CA47234 (to M. Z. H.) * This work was supported in part by American Cancer Society Grant

National Institutes of Health Grant ES05694 (to J. J. S.), and National Institutes of Health center Grants ES00260 and CA16087. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, UMD-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103-2714. Tel201-982-5217; Fax: 201-982- 3644; E-mail: [email protected].

tion fork to force error-prone replication past such sites. More recent experimental evidence has been interpreted to mean that SOS-mutagenesis results from the combined action of the products of three SOS genes, namely r e d , u m u D , and umuC (11-17). The RecA protein (in its activated “RecA*” form) in particular is hypothesized to play a crucial role in SOS mutagenesis because, in addition to its direct role at the stalled replication site, RecA* is required for the derepression of the SOS regulon, and for the proteolytic activation of UmuD to UmuD’. These three SOS proteins are believed to be responsible for both of the proposed steps for SOS mutagenesis, namely, base incorporation opposite a noninstructive site, and lesion bypass (18-23).

The SOS hypothesis successfully integrates a number of observations regarding cellular responses to DNA damaging treatments, and in particular accounts for Weigle reactivation and Weigle mutagenesis, two phenomena that refer to en- hanced survival and mutagenesis of UV-irradiated phage A in UV-irradiated Escherichia coli cells as compared to non-irradiated cells (24). However, the SOS hypothesis, as it appears to be currently interpreted in the literature, suffers from a few inconsistencies. First, error-prone replication has not been unambiguously demonstrated to depend on the addition of purified RecA*, UmuD’, and UmuC proteins to defined in vitro replication systems using the DNA polymerase I11 complex. Second, defects in umuD and umuC genes result in slight, but not profound sensitivity to DNA damaging treatments. This observation is difficult t o reconcile with their proposed critical role at the stalled replication fork, whether it is in base incorporation opposite a lesion in the absence of instruction or elongation in the absence of a correctly paired 3‘ terminus. These inconsistencies, as well as other considerations, leave open the possibility that additional, as yet unrecognized, factors may be involved in the mutagenic bypass of noninstructive lesions.

3,N4-Ethenocytosine (&)I is a mutagenic exocyclic DNA lesion induced by metabolites of industrial chemicals such as vinyl chloride (25) and ethyl carbamate (26). Recent work has demonstrated that EC has the i n vitro and in vivo template properties expected of noninstructive DNA lesion (27-311, a finding that is consistent with the fact that two of the three

3,N4-ethenodeoxycytidine; DBU, 1,8-diazabicyclo[5,4,OJ-undec-7-ene; ’ The abbreviations used are: EC, 3,N4-ethenocytosine; EdCyd,

ssDNA, single-stranded DNA, ssDNA,, ssDNA construct bearing a single, site-specific EC residue; ssDNA,-, a control ssDNA construct prepared by the same procedures as for ssDNA, except that a normal cytosine replaces the EC residue; gDNA, circular gapped duplex DNA pol I, pol 11, and pol 111, E. coli DNA polymerases I, 11, and 111, respectively; DMTr-EdCyd, 3,N4-etheno-5’-0-(4,4‘-dimethoxytrityl)-2’-deoxycytidine; DMTr-EdCyd-phosphoramidite, 3,N4-etheno-5‘-0-(4,4’-dimethoxytrityl)-2‘-deoxycytidine-3’-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel; HPLC, high performance liquid chromatography; nt, nucleotide; UVM, W, modulation of mutagenesis.

27433

27434 SOS-independent Inducible Mutagenesis Watson-Crick hydrogen-bonding positions are bridged over in EC. EC residues within short (17 or 174 nt) single-stranded "gaps" in circular double-stranded M13 DNAmolecules are pro- foundly mutagenic in both r e d + and in r e d - cells without appreciably affecting survival (27, 28, 30, 31). These findings suggest that replication past noninstructive EC lesions during gap-filling DNA synthesis does not require SOS functions. However, prior UV irradiation of both red' and r e d - (ArecA) cells brings about a pronounced modulation of mutagenesis at an EC residue borne within the gap, indicating the existence of an SOS-independent inducible mutagenic phenomenon. We have named this phenomenon UVM mutagenesis for UVmodu- lation of mutagenesis.

Since gap-filling DNA synthesis can in principle be catalyzed by DNA polymerase I, 11, or 111, and since protein co-factor requirements for gap-filling can differ from those for "normal" DNA replication, mutation furation opposite a lesion during gap-filling may differ from that occurring during normal replication. To test whether UVM is strictly a phenomenon of gap- filling synthesis or whether it is also observable during replication by DNA polymerase 111, we have examined mutagenesis at an EC residue borne on M13 viral single-stranded DNA (ssDNA). In the M13 replication cycle, the viral ssDNA is con- verted to a double-stranded replicative form DNA by initiation of replication at a unique site (32). The initial primer, synthesized by E. coli RNA polymerase, is elongated around the circle by DNA polymerase 111. By analyzing mutation frequency and specificity using a multiplex sequence assay, we show here that UVM is observable in UV irradiated r e d + , and in irradiated ArecA cells. These data imply that UV irradiation induces a previously unrecognized mutagenic mechanism in E. coli, and that this mechanism can operate during gap-filling synthesis as well as during normal DNA replication.

EXPERIMENTAL PROCEDURES Materials and Bacterial Strains-Deoxycytidine was purchased from

Sigma. Chloroacetaldehyde (50% w/v aqueous solution), silica gel (Merck, grade 60,230-400 mesh, 60 A), 1,8-diazabicyclo[5,4,0lundec-7- ene (DBU), N,N-diisopropylamine, 4,4'-dimethoxytrityl chloride, anhydrous pyridine, Drierite, and Maxam-Gilbert DNA sequencing reagents were obtained from Aldrich. Ultrapure electrophoresis reagents were purchased from Bio-Rad or IBI. Fluorescent thin layer chromatography (TLC) plates (Silica Gel 60 F,,,; prepared by EM Science) were obtained from VWR Scientific. Chloro(N,N-diisopropylamino)(cyanoethoxy) phosphine and 4-A molecular sieves were purchased from Fisher Sci- entific. Diisopropylamine, methanol, and other high purity solvents were dried by distillation from calcium hydride and stored over molecular sieves. All other chemicals were high grade quality obtained from various vendors. Silica gel column chromatography was performed in the presence of 0.1% pyridine. TLC plates were developed in the following solvents: I, methano1:chloroform (15:85); 11, methano1:chloroform (10:90); 111, benzene:acetone (30:70); IV, triethy1amine:ethyl acetate: chloroform (10:45:45). T4 DNA ligase and T4 polynucleotide kinase were purchased from New England Biolabs; Sequenase Version 2.0 was from U. S. Biochemical Corp. Deoxynucleoside triphosphates and dideoxynucleoside triphosphates were from U. S. Biochemical Corp. or Pharmacia Biotech Inc. Except for the &-containing oligonucleotides and the corresponding controls, all other oligonucleotides were prepared by conventional automated procedures and purified by denaturing polyacrylamide gel electrophoresis before use. The E. coli strains KH2 r e d ' and KH2R A r e d have been described previously (33). Phage M13 L2 (34) was a gift of Dr. Christopher Lawrence.

Synthesis of 3,N4-Ethenodeoxycytidine (EdCyd; Fig. 1)"edCyd was synthesized by the following modifications of the procedures previously described (35). Deoxycytidine (4 mmol) was reacted with 40 ml of a 2 M

aqueous solution of chloroacetaldehyde at 37 "C and pH 3.5 with shaking. The pH of the reaction mix was monitored and adjusted to pH 3.5 with 1 N NaOH at regular intervals (every 30 min at the initial stage and at approximately 120-min intervals at late stages). The formation of EdCyd was followed by TLC, using solvent I. The reaction, which was usually about 90% complete by 16 h, was terminated by removal of unreacted chloroacetaldehyde by concentration under reduced pres-

N - l

I

Q R2

R1 = H or 4,4'-dimethoxytrityl

R2 = H or (2-cyanoethyl)-N,N diisopropyl-phosphoramidite

FIG. 1. Structure of edCyd and derivatives. EdCyd: R l = R2 = H. DMTr-EdCyd: R l = DM-, R2 = H. DMTr-EdCyd-phosphoramidite: R l = DMTr; R2 = (2-cyanoethyl)-N,N- diisopropylphosphoramidite.

sure. The residue was dried by repeated (3 x 50 ml) co-evaporation with toluene and the toluene in turn was removed by co-evaporation with methanol (2 x 100 ml). The crude EdCyd, dissolved in 8 ml of methanol: chloroform (5:95), was purified by chromatography on a silica gel column (2 x 30 cm) preequilibrated with methano1:chloroform and a trace (0.1%) of pyridine. EdCyd was eluted with a 5-10% methanol gradient in chloroform. Fractions (7 my5 min) containing the purified cdCyd were detected by TLC in solvent I, pooled, and concentrated under reduced pressure. The product, dissolved in 6 ml of methano1:chloroform (5:95), was precipitated from petroleum ether (300 ml). The precipitate was collected by centrifugation at 2500 rpm in a desktop centrifuge for 5 min a t room temperature, dried under vacuum, and stored at -20 "C over a desiccating agent (Drierite). The yield was 60%, and the product was homogeneous as judged by a single spot on TLC. The R, values were: solvent I, 0.25; solvent 11, 0.12; and solvent 111, 0.16. The product was free from the starting material deoxycytidine, which had the RF values: solvent I, 0.054; solvent 11, 0.018; and solvent 111, 0.02.

Characterization of EdCyd-EdCyd was characterized by W, mass spectrometry, and NMR. The W spectrum was identical to that previously reported (36). The W spectra at pH 1 (1 N HCl), 6.3 (H,O) gave A,, a t 289 and 270 nm, respectively. The electron impact mass spectrum was similar to the spectrum previously reported (25). The isobu- tane chemical ionization (chlorine) mass spectrum established a molecular weight of 251. The chemical shift (6) assignments in the 'H NMR of EdCyd (360 MHz; D6 - MeSO,) was as follows: 7.80 and 7.39 (2s,2H, etheno), 7.74 (d, lH, J = 8.0 Hz, H6), 6.74 (d, lH, J = 8.0 Hz, H5), 6.42

J = 5.1 Hz, 5'-OH), 4.30 (m, lH, H3'), 3.86 (q, lH , H4'), 3.62 (m, 2H, H5'), 2.21 (m, 2H, H2'). These assignments were checked by two di- mensional correlated spectroscopy NMR. The structure was unambiguously established by these spectroscopic methods as edCyd.

Preparation of 3,N4-Etheno-5'-O-~4,4'-dimethoxytrityl)-2'-deoxycytidine (DMIF-edCyd)--EdCyd (8 mmol) was tritylated at the 5'-OH by reaction with 4,4'-dimethoxytrityl chloride in anhydrous pyridine (37). The crude DMTr-edCyd was purified by silica gel chromatography as follows. The product, dissolved in 20 ml of chloroform, was applied to a silica gel column (2.5 x 30 cm) preequilibrated with chloroform containing a trace (0.1%) of pyridine, and eluted with a 1 4 % methanol gradient in chloroform (spiked with 0.1% pyridine). Fractions (10 my7 min) containing pure DMTr-EdCyd (single spot on TLC in solvent 11) were pooled and the product recovered as described above for EdCyd. DMTr- EdCyd (yield, 85%) was homogeneous as judged by a single spot on TLC, had the following R, values: solvent 11, 0.57; solvent 111, 0.71; and solvent N, 0.35. I t was free of the starting material EdCyd whose RF values were: solvent 11, 0.12; solvent 111, 0.16; and solvent IV, 0.01.

Characterization ofDMWEdCyd-The structure of DMTr-EdCyd was elucidated by mass spectrometry and NMR. The chlorine mass spectrum indicated a molecular weight of 553. The 'H NMR of DMTr - EdCyd (360 MHz, CDCl,) was assigned as follows: 7.71 and 7.36 (2% 2H, etheno), 7.62 (d, lH, J = 8.0 Hz, H6), 7.43 - 6.80 (m, 13H, aromatic protons of DMTr), 6.59 (t, lH, J = 6.2 Hz, Hl ') , 6.35 (d, lH, J = 8.0 Hz, H5), 4.61 (m, lH, H3'), 4.07 (m, lH, H4'), 3.79 ( s , 6H, OCH,), 3.50 (m, 2H, H5'), 2.51 (m, lH, H2'-P), 2.35 (m, lH, H2'-a).

Preparation of 3,N4-Etheno-5'-0-(4, 4'-dimethoxytrityU-2'-deoxycytidine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel (DMlY- EdCyd-ph0sphoramidite)-Published procedures (381, as modified for the synthesis of the 3'-phosphoramidite of DMTr-(NB-Et)dThd (39) and

(t, lH, J = 6.7 HZ, Hl ') , 5.33 (d, lH, J = 4.2 HZ, 3"OH), 5.10 (t, lH,

SOS-independent Inducible Mutagenesis 27435

U 0

s

FIG. 2. HPLC analysis of snake venom phosphodiesterase and alkaline phosphatase digests of control or &-containing 17-mer on an HP Hypersil ODS column using an acetonitrile gradient ( 0 5 % ) in 10 mM potassium phosphate, p H 4.5 (29).

of DMTr402-Et)dThd (40) were applied to 1.8 mmol of DMTr-EdCyd. The crude DMTr-EdCyd- phosphoramidite was purified by silica gel column chromatography as described (391, and the purified material was precipitated from dry hexane a t -80 "C. The precipitate was dried over phosphorous pentoxide under vacuum, and aliquoted into glass vials for storage at -80 "C over a desiccating agent (yield, 73%). The pure DM*-EdCyd phosphoramidite (formula weight: 769) gave two closely moving spots on TLC indicating the presence of diastereomers. The R, values were: solvent III,0.85 and 0.82; solvent N, 0.66 and 0.63. The amidite was free from the starting material, DMTr-EdCyd, whose R, values were: solvent 111, 0.71; solvent N, 0.35.

Chemical Synthesis of Oligodeoxynucleotides with Site-specific cdCyd-A 17-mer (5'-ATGAACXGGAGGCCCAT; X = EC), was synthesized by conventional automated methods on an Applied Biosystems model 380A synthesizer except that the operation was stopped after the last detritylation step, and subsequent steps were carried out manually. Mild non-aqueous deprotection and cleavage from the silica matrix were carried out using DBU in anhydrous methanol. The silica matrix was transferred to a 50-ml round-bottom flask and dried by repeated co- evaporation with dry toluene (3 x 15 ml). Seven ml of a mix containing 1 ml of DBU and 6 ml of dry methanol were transferred to the flask under anhydrous conditions, and the contents was incubated for 14 days a t room temperature with occasional shaking (40). The silica sus- pension was centrifuged to recover the supernatant. The silica pellet was extracted with 0.5-ml portions of water thrice. The supernatant and the three extracts were pooled and repeatedly extracted with benzene (5 x 5 ml) to remove DBU. The pH was adjusted to 6.5 with 80% acetic acid, and the material was concentrated under reduced pressure. The crude oligomer was desalted on a Sephadex G-50 column (1 x 50 cm) using ethanokwater (20:80) as the eluent. The oligomer-containing fractions were concentrated, and the oligomer was purified by gel electrophoresis on 16% polyacrylamide, 8 M urea gels.

HPLC Analysis of Nuclease Digests-The synthetic oligonucleotides containing EC and corresponding control oligomers were digested with snake venom phosphodiesterase and calf intestinal alkaline phosphatase as described previously (29) and analyzed on a C-18 column (HP hypersil ODs) as described elsewhere (29). Analysis of &-oligomer digests (but not controls) revealed a peak corresponding to EdCyd (Fig. 2) in the expected molar proportion to the four normal bases. The EdCyd peak was unambiguously identified by coelution with an authentic marker and by W absorption characteristics.

Sequence Analysis and Detection of Alkali-labile Site-The oligonucleotides were 5' end-labeled with 32P and subjected to Maxam-Gil- bert sequence analysis as described elsewhere (29). This analysis con- firmed the sequence and revealed the presence of a single alkali-labile site coinciding with the expected position of EC (Fig. 3).

Construction of M13 Single-stranded DNA Bearing a Site-specific cC-Procedures previously described for U V photoproducts (41, 42) were used for constructing single-stranded DNA containing a site-specific aC residue (ssDNA,) or the corresponding control DNA construct (ssDN&) bearing normal cytosine in place of EC, as outlined in Fig. 4.

FIG. 3. Maxam-Gilbert DNAsequence analysis of &containing

lability of EC, all lanes show a strong band a t position X for the lesion- 17-mer or control oligonucleotide. As expected, due to the alkali-

bearing 17-mer (X = cC), but not for the control (X = C). Also note that the control oligonucleotide, but not the &-bearing 17-mer. has an area of band compression (top third of the ladder) presumably due to an unidentified secondary structure.

17-mer

57-mer

FIG. 4. Strategy for construction of ssDNA,. The procedures of Lawrence and co-workers (41, 42), as described under "Experimental Procedures," are outlined at the top. At the bottom, DNA sequences of the 3' and 5' ends of the linearized ssDNA, of the 17-mer, and of the 57-mer "scaffold" are given. In addition, a synthetic "anti-scaffold" 57- mer with a sequence complementary to that of the scaffold was used for obtaining molecule 4 from molecule 3 as described under "Experimental Procedures."

Phage M13mp7L2 (34) was grown in E. coli KH2R (Sup' Are&) strain, followed by phage particle purification and ssDNA extraction as described elsewhere (43). Approximately 800 pg of purified ssDNA was linearized by digestion with about 4000 vendor units of EcoRI (New England Biolabs) for 1 h in 1 ml of buffer (50 mM NaCI, 100 mM Tris- HCl, pH 7.5, 5 mM MgCl,, and 0.05% Triton X-100, supplied as a 10 x concentrate by the vendor) at 37 "C. A fraction of the digest was examined by electrophoresis in 1.4% agarose gels in TBE buffer (50 mM Tris-borate, 10 mM EDTA, pH 8.3) to verify complete linearization resulting in a faster moving band. The linear ssDNA (10 pglml) was annealed with a 2-fold molar excess of a 57-mer "scaffold" (see Fig. 4 for sequences) in buffer (50 mM Tris-HC1, pH 7.8,lO mM MgCl,, and 20 mM dithiothreitol) by heating at 90 "C for 3 min followed by slow cooling to 50 "C over a period of 2-3 h. Because the 5' 20 nt and the 3' 20 nt of the 57-mer are complementary, respectively, to the 3' and 5' ends of the linear viral DNA, annealing of the two DNAs generates a circular ssDNA species with a 17 nt gap in the viral DNA strand. A ten fold molar excess (over ssDNA) of a 17-mer with a sequence complementary to the central 17 n t of the scaffolding 57-mer, bearing a single EC residue (or cytosine in the case of the control construct) was added to the annealing mix held at 50 "C, and the contents were allowed to slow cool to 16 "C over a period of 3 4 h. Ligation was initiated by the sequential

27436 SOS-independent Inducible Mutagenesis addition of ATP to a final concentration of 1 mM, and T4 DNA ligase (New England Biolabs) to a concentration of 200 vendor unitdml. The contents were incubated at 16 "C for 16 h. An aliquot of the ligated DNA was denatured by heating at 90 "C for 5 min in the presence of 10-fold excess (over ssDNA) of an "anti-scaffold" oligonucleotide (a 57-mer with a sequence complementary to the scaffolding 57-mer). Agarose gel anal-

- G C T A T G A C C A T G A T T C A G T G A T ~ ~ ~ ~ C C A T M T T C A ~ ~ C G T C G ~ T T - TCCGGGTATTMGTGACCG-5"

Mg++ Sequenase 2.0

-hcGcGGcGc= Primer Elongation Products

f \ (Wild typa - 24 n t )

- A C G G -

(-1 n t deletion - 231ner) m G C C -

- A C A G G - m c c - - 2':

C-to-A transversion - 22-mer) - 21 - 22

- A C T G G - c c - = - 19

(C-to-T t ransi t ion - 21 n t

- A C C G G - 1 (Unelongated Primer - 19 n t )

FIG. 5. Strategy for determining frequency and specificity of mutagenesis at a site-specific rC residue borne on M13 ssDNA. Pooled progeny phage DNA, consisting of wild type DNA(N = C), C-to-T transitions (N = T), C-to-A transversions (N = A) or 1-nt deletions (-1) was annealed to a 5' end-labeled (3zP) primer and incubated with Se- quenase 2.0 (T7 DNApolymerase defective in 3'4' exonuclease activity) in the presence of two normal nucleotide precursors (dCTP, dGTP), a chain-terminating precursor (ddTTP), and M e . Under these conditions, limited primer extension occurs such that elongation on each of the above four species of template DNA results in a product of different length. The proportion of each of the three mutants is determined by computing densitometric analysis of the three mutant bands (21-, 22-, and 23-mer) as described under "Experimental Procedures." Experi- ments with artificial mixes of wild type, C-to-T, and C-to-A DNAs (Figs. 6 and 7) show that the strategy can measure the frequency of each mutation with reasonable accuracy. It should be noted that C-to-G transversions, assayed by a similar strategy, are not induced by Ec at significant levels (not shown).

-

ysis revealed that ligation was about 50% complete. Procedures for ssDNA Dansfection, and Preparation of Pooled Prog-

eny Phage DNA-Procedures for transfection into non-irradiated and UV-irradiated E. coli cells, for measurement of survival (transfection efficiency), and for the preparation of pooled progeny phage DNA have been described previously (30, 31, 44).

Quantitative Multiplex Sequence Analysis of Pooled Progeny Phage DNA-The principles and experimental validation of the multiplex assay have been previously described (31,44). The sequence of the template, and the specific primer and nucleotide triphosphate combinations used for the experiments described here are depicted in Fig. 5. Mutation frequency is derived from the normalized densitometric signal in the mutant bands (21-mer, 22-mer plus 23-mer), as follows.

Mutation frequency = [21-mer]+[22-mer]+[23-mer]

[21-mer]+[22-mer]+[23-mer]+[24-mer]' (Eq. 1)

The individual frequencies for C-to-T transitions, C-to-A transversions and -1 nt deletions (i.e. mutational specificity) is similarly determined from the normalized signal in the corresponding band as follows.

C-to-T transitions = [21-mer]

[21-merl+[22-merl[+[23-merl+[24-merl (Eq. 2)

C-to-A transversions = [22-merl

[21-merl+[22-merl+[23-mer]+[24-mer] (Eq. 3)

- 1-nt deletions = [23-merI

[21-merl+[22-merl+[23-merl+[24-merl (Eq. 4)

A template-primer DNA complex was prepared by annealing 2 pmol of pooled progeny phage viral ssDNA (template) with 1 pmol of a 5 ' - 32P-end labeled 19-mer primer as described previously (44). In each reaction, 0.2 pmol of the annealed DNA complex (i.e. 0.2 pmol of template, 0.1 pmol of the primer) was incubated in 10 1.11 of buffer (50 mM Tris-HCI, pH 7.8, 10 mM MgCI,, and 20 mM dithiothreitol) containing 1 PM dCTP and 1 PM dGTP, 0.05 PM of ddTTP, and 0.1 unit of Seque- nase Version 2.0 (a T7 DNA polymerase derivative devoid of 3'40-5' editing exonuclease activity) for 15 min at 37 "C. The reaction was terminated and the products analyzed by high resolution denaturing polyacrylamide gel electrophoresis, followed by densitometric analysis of the autoradiographs as described previously (44). Fig. 6 shows an analysis of a series of DNA preparations created by mixing authentic

A T 1 C G

CIAIT G T G c c c G c c c A T T T $ : ! $

" - - n 4 "

F = E

e e :z 5 2 5 2

?. 2 0

: $ : w m

3 S G !-

The "mutant DNA" is an equimolar mixture of C + A transversions and C + T transitions. Mutant DNA was added to the wild type to simulate FIG. 6. Multiplex sequence analysis of mixtures of wild type and mutant DNAs for verification of the strategy outlined in Fig. 5.

the following mutation frequencies: lane 1, 0%; lane 2,2.5%; lane 3, 5%; lane 4, 10%; lane 5,20%; lane 6,30%; lane 7,40%; lane 8, 50%; and lune 9,60% (percent figures represent total mutant frequencies; e.g. the DNA mix in lane 4 has the following composition: wild type, 90%; C "-f A, 5%; C + T, 5%). I t can be seen that, as the mutant fraction increases, there is an orderly increase in the intensity of the "mutant bands" (21- and 22-mer) at the expense of the wild type band (24-mer). Results of a quantitative analysis of such autoradiographs are shown in Fig. 7.

SOS-independent Inducible Mutagenesis 2 743 7

0 10 20 30 40 50 60 Expected Mutation Frequency

FIG. 7. Plot of experimentally determined "mutation frequencies" against expected frequencies to demonstrate that the multiplex sequencing strategy outlined in Fig. 5 can estimate mutation frequencies with reasonable accuracy. Autoradiographs such as the one shown in Fig. 6 were analyzed by computing densito- metry. Each data point represents an average of four separate elongation reactions.

wild type DNA (where N = C) with increasing proportions of two mutant DNAs (N = T; N = A). Fig. 7 is a plot of the "mutational frequencies" deduced from experiments, such as the one shown in Fig. 6, and shows that the assay allows the quantitative detection of major mutational events with reasonable accuracy. It may be noted that in this particular assay, any C-to-G transversions will give rise to a 24-mer, and therefore will not be separable from the wild type. However, applying a specific assay for detecting C-to-G transversions did not show significant levels of C-to-G transversions detectable by multiplex assays. This finding is in agreement with previous clone-by-clone sequencing results showing that C-to-G transversions are not induced at significant levels by EC (data not shown).

RESULTS

The Experimental System-The experimental system con- sists of (i) construction of MI3 ssDNA bearing a site-specific EC residue (ssDNA,) by procedures outlined in Fig. 4, (ii) transfection of ssDNA,, into non-irradiated or UV-irradiated E. coli host cells, (iii) plating of infectious centers to determine survival effects, and (iv) DNAsequence analysis of mutant progeny for determining the mutagenic consequences. Mutagenesis of the viral progeny resulting from the in vivo replication of ssDNA,, was determined by using a quantitative multiplex sequence analysis assay (441, as described in Figs. 5-7, a technology that allows a high resolution analysis of the effects of variables on mutational hot spots by rapid and convenient procedures.

Effect of a Site-specific EC Residue on Survival of MI3 Single- stranded DNA Genome-Table I shows that when ssDNA,, is transfected into unirradiated cells, survival drops to approximately 11% in red' cells, and about 17% in A r e d cells. UV- irradiation of cells before transfection increases the apparent survival of ssDNA,,, such that at a UV dose of 25 J/m2, survival is around 45% in red' cells, and about 28% in A r e d cells.

Effect of W Irradiation of red' E. coli Cells on the Fre- quency and Specificity of Mutagenesis Opposite &-Fig. 8 shows that in non-irradiated r e d + cells, mutagenesis at the site-specific EC residue is low but detectable. UV-irradiation of cells before transfection of ssDNA, results in a dramatic enhancement in mutagenesis at the EC residue. Table I1 summarizes data from two independent series of transfections and six multiplex assays, and allows the following conclusions. UV- irradiation of red' cells before transfection of ssDNA, results in a W dose-dependent increase mutation frequency from around 4% to over 50%. Both C-to-T transitions and C-to-A

TABLE I The effect of prior W irradiation of recA' and ArecA cells on the

survival of ssDNA,, and ssDNA,

W to Cells Exp::yt DNA construct red' cells Are& cells ~

2.5

25

5

10

JI m2 0 I

I1

I

I1

I

I1

I

I1

I

I1

I

I1

50

Reference" ssDNA, ssDNA,, Reference ssDNA, ssDNA,,

ssDNA, ssDNA,,

ssDNA, ssDNAec

ssDNA, ssDNAe,

ssDNA, ssDNA,,

ssDNA, ssDNA,,

ssDNA, ssDNA,,

ssDNA, ssDNAz,

ssDNA, ssDNAec

ssDNA, ssDNAec

ssDNA, ssDNA,,

pfub/50 ng 8587 2270 (100)' 1127 (100)

2497

343 (15) 210 (19) 3060 2693 (100) 1650 (100)

1803

187 (7) 250 (15)

NDd 690 (100) ND 170 (25)

ND 1190 (100) ND 183 (15)

1433 (100) 517 (100) 457 (32) 147 (28)

2967 (100) 583 (100) 833 (28) 170 (29)

3427 (100) 417 (100) 1613 (47) 150 (36)

3753 (100) 853 (100) 1313 (35) 260 (30)

3227 (100) 506 (100) 1273 (39) 143 (28)

3993 (100) 783 (100) 2040 (51) 133 (27)

3253 (100) ND 1743 (54) ND

1633 (100) ND 1477 (90) ND

e A preparation of MI3 L2 ssDNA used as a reference for transfection efficiency.

Numbers in parentheses are percent of control. Plaque forming units.

ND, not determined.

transversions are about equally represented and together account for almost all of the mutagenesis, with -1 nt deletions constituting a minor fraction.

Effect of W Irradiation of Ared E. coli on Mutagenesis Op- posite &-Fig. 9 demonstrates that UV irradiation of RecA- deleted E. coli with relatively low UV doses before transfection results in a stimulation of mutagenesis opposite EC residues. Table I11 summarizes data from two independent series of transfections and six multiplex assays, and permits the following conclusions. In non-irradiated A r e d cells, significant mutagenesis occurs opposite the EC residue. W irradiation of these cells before transfection results in a UV dose-dependent increase in mutagenesis to about 60%. In non-irradiated cells, most mutations appear to be -1 nt deletions, with C-to-T transitions and C-to-A transversions accounting for a minor fraction. Prior W irradiation of host cells appears to dramatically enhance base substitutions at the cost of -1 nt deletions,

DISCUSSION

The molecular processes responsible for mutation fixation opposite noninstructive lesions have attracted a great deal of interest because a majority of extrinsic and intrinsic mutagens inflict noninstructive rather than mispairing lesions in DNA. In seeking a theoretical framework for describing the underlying mechanisms, we have hitherto relied almost exclusively upon the SOS hypothesis. Palejwala et al. (31) have recently

27438 SOS-independent Inducible Mutagenesis

UV Dose ( J l d

0 5.0 10 25 50 ““P

C E C c E C c &C c &C c E C FIG. 8. Multiplex DNA sequence analysis for determining mu-

tation frequency and specificity of pooled progeny phage DNA obtained by transfection of ssDNA, (lanes labeled EC) or the control construct (lanes labeled C) into UV-irradiated E. coli KH2 cells (recA’ cells). Pooled progeny phage DNA was isolated from each transfection and the multiplex assay was carried out as described in Fig. 5 and under “Experimental Procedures.” A 5’ 32P-end-labeled 19-mer primer annealed to progeny pool DNA was elongated by T7 DNA polymerase (Sequenase Version 2.0; U. S. Biochemical Corp.) in the presence of dGTP, dCTP, and ddlTP. The numbers on the right identify the lengths of elongation products. In this assay, the wild type template yields a 24-mer. C -+ T transition yields a 21-mer, a C -, A transversion template yields a 22-mer, and -1 mutation gives rise to a 23-mer. The DNA sequence of the template in the vicinity of the primer 3”OH terminus is as shown in Fig. 5. All progeny obtained by transfection of the control construct is wild type (lanes C). In contrast, transfection of EC containing ssDNA (lanes eC) results in wild type (band 24) as well as mutant (bands 21-23) progeny. The mutation frequency at EC is low in unirradiated cells but increases as a function of prior UV irradiation

Table 11. of host cells. These observations are expressed quantitatively in

TABLE I1 Effect of prior UV irradiation of red’ E. coli on mutagenesis a t a

site-specific EC residue on M13 ssDNA

W to cells Mutation frequency Mutation specificity

C + T C - A - l n t

J lm2 % (d3.D.”) % ( 4 . D . ) 0 4 (22) 3(t1) 1(+0) <1 5 18 (+6) 10

5 (22) 8(+3) 5 ( t4) 31 (+7) 12(+4) 17(+4) l (4 )

25 50

56 (+9) 22 (+6) 34(+3) <1 51 ( t7) 13( r4) 37(+3) l(t1)

Averages derived by analyzing progeny DNA pools obtained in two separate transfection experiments, with each pool subjected to three multiplex sequencing assays. S.D. numbers were rounded to the nearest integer.

raised the possibility that UVM may represent a parallel, previously unrecognized component of inducible cellular mechanisms responsible for mutagenic processing of DNA lesions.

The major question addressed here concerns whether UVM is associated strictly with gap-filling DNA synthesis or whether it is also manifested during normal DNA replication. Results described in this study establish that UVM is also manifested during DNA synthesis catalyzed by E. coli DNA polymerase 111, the major replicative enzyme responsible for leading strand synthesis as well as for a major part of the lagging strand synthesis in E. coli. These results also render unlikely several alternative explanations (e.g. a sequence context effect) that could have accounted for the original observations (311, and suggest that UVM is a potentially significant inducible mutagenic phenomenon.

Fig. 10 summarizes the principal observations on UVM mutagenesis a t site-specific EC residues in ssDNA and in circular gapped duplex DNA (gDNA). It is interesting that even though the final magnitude of the UVM effect is similar in the two constructs (about 60% of progeny is mutant), constitutive mutagenesis is lower in ssDNA in red’ host cells (-4%) as com-

0

UV Dose ( J l d

5.0 ”

”

0 . 0 ”

10.0 25.0

24 23 22 21

c E C c E C c E C c EC FIG. 9. Multiplex DNA sequence analysis for determining mu-

tation frequency and specificity of pooled progeny phage DNA obtained by transfection of ssDNA, (lanes labeled cC) or the control construct (lanes labeled C) into UV-irradiated E. coli KH2R recA- cells. The assays were carried out as described in the legends for Figs. 5 and 8 and under “Experimental Procedures.” It can be seen that mutation frequency increases as a function of prior W irradiation of cells. These results are expressed quantitatively in Table 111.

TABLE I11 Effect of prior W irradiation of ArecA E. coli on mutagenesis a t a

site-specific EC residue on M13 SSDNA

W to cells Mutation Frequency Mutation specificity YO

C - T C - A - l n t

Jlm2 % (A3.D.”) (tS.D.) 0 13 (+3) l(t1) 1(+1) l l ( t3 ) 5 33 (t3) 21(+3) 12 ( t3 ) 1k1) 10 48 (k7) 18(+4) 30(+4) <1 25 60 (=2) 30(+2) 30(+3) l(t1)

Averages derived by analyzing progeny DNA pools obtained in two separate transfection experiments, with each pool subjected to three multiplex sequencing assays. S.D. numbers were rounded to the nearest integer.

40 Gapped Duplex DNA Single-Stranded DNA

3 $ $ E L

3 E

3 E

4 E

FIG. 10. Comparison of the UVM effect in gDNA (data from Ref. 31) and in ssDNA, (data from this work). The strains identified as recA+ and recA- are, respectively, E. coli KH2 red’ and E. coli KH2R red-. Strains identified as recA+(UVj or recA-(W) are, respectively, KH2 re& or KH2R r e d - subjected to a W dose of 25 J/m2 before transfection.

pared to gDNA (-25%). The lower frequency in the ssDNA construct is in agreement with results from other laboratories that have also used site-specific adduction technology to inves- tigate EC mutagenesis (45).

The difference in constitutive mutagenesis a t EC residues in ssDNA and in gDNA may reflect a difference at the level of

SOS-independent Inducible Mutagenesis 27439

rn C

250 determine whether this represents Weigle reactivation, because a reactivation phenomenon, albeit of a lower magnitude,

cannot occur according to the SOS hypothesis. This latter re-

.- L m also occurs in A r e 4 cells (Fig. 11) in which Weigle reactivation 0 200 0

P activation is reminiscent, in its r e d - independence, of a similar al! reactivation previously reported for 4x174 replica form DNA 0 0 ' 5 0 bearing non-mutagenic thymine derivatives replicative (thy- g5 mine glycols) (49).

p 5 100 '5

$ 0

(c '5

0 - In conclusion, the results presented here confirm and extend the original observations (31) that UVM, an SOS-independent inducible phenomenon in E. coli, modulates mutation-fixation, error-avoidance and bypass at noninstructive lesions. Under- standing the mechanisms underlying UVM must await further characterization of the phenomenon.

Acknowledgments-V. A. P. and G. A. P. made an equal contribution to this work. We wish to thank Dr. Christopher Lawrence for phage M13 mp7L2 and Rehan Ahmad for conscientious technical assistance.

u)

$ 50 .- * m - 3 3 5

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