The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA

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Mutation Research, 249 (1991) 189-193 © 1991 ElsevierSciencePublishers B.V. 0027-5107/91/$03.50 ADONIS 0027510791001406

MUT 04992

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA Grigory L. Dianov, Murat K. Saparbaev, Alexander V. Mazin and Rudolf I. Salganik Institute of Cytology and Genetics, U.S.S.R. Academy of Sciences, Siberian Branch, Nooosibirsk 630090 (U. S. S.R.)

(Received 10 September1990) (Revision received14 December1990) (Accepted 7 January 1991)

Keywords: Plasmid recombination,mutagen-stimulated; Dimethylsulphate; Uracil; RecA binding

Summary The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid D N A induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to D N A . We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.

The frequency of homologous DNA recombination in various organisms can be increased by treatment with either chemical mutagens or UV radiation (Jacob and Wollman, 1953; HowardFlanders et al., 1968; Curtiss, 1968; Baker and Haynes, 1972; Taylor et al., 1983; Abbott, 1985a). It was proposed that mutagenic treatment stimulates the repair of damaged D N A and that repair intermediates such as nicked or gapped molecules are able to initiate homologous recombination

Correspondence: Dr. Grigory L. Dianov (present address), Imperial Cancer Research Fund, Clare Hall Laboratories,South Mimms, Potters Bar, Herts. EN6 3LD (Great Britain).

(Baker and Haynes, 1967; Howard-Flanders and Boyce, 1966; Smith and Hays, 1985). However, several experimental observations contradict this mechanism: (i) Mutations in repair genes do not reduce the level of the induced recombination (Abbott, 1985b; Smith and Hays, 1985); (ii) Excision repair of uracil residues (Hays et al., 1981) or repair of single-base mismatches (Lieb, 1985) has little or no effect on recombination. To examine the mechanism of mutagen induced recombination and the role of D N A repair in this process, we have developed an in vivo recombination assay based on plasmids derived from pBR322 and containing non-overlapping mutations in the tet gene. Co-transformation of plasmids into E.

190

coli led to recombination between molecules of plasmid DNA and thereby gave rise to the tetracycline-resistant bacterial colonies. We have employed two different types of plasmid DNA altered by DMS treatment or uracil incorporation, used these DNAs in the recombination assay and demonstrated that DMS-treated D N A is more efficient in stimulation of recombination than uracil-containing DNA. By in vitro studies with purified RecA protein we have shown that DMS treatment of plasmid DNA, but not uracil incorporation, stimulates RecA protein binding to damaged DNA. We hypothesize that induction of recombination by chemical mutagens is initiated by activation of the first step of recombination: RecA protein binding to DNA and formation of heteroduplex molecules. Materials and methods

Microbiological techniques. Bacterial strains and plasmids used in this study are listed in Table 1 or described in the text. Media and general growth conditions were those described by Maniatis et al. (1982). D N A isolation and manipulation. Plasmid DNA was isolated from E. coli ABl157 or HB101 as described by Clewel and Helinski (1969). Plasmid DNA containing about 10% of the thymidine residues substituted by uracil (uracilcontaining-DNA) was isolated from E, coli RZ1032 (Sagher and Strauss, 1983).

D M S treatment was performed in a buffer containing 50 mM sodium cacodilate (pH 8.0), 1 mM EDTA, 1% DMS at 23°C for 5 min. D N A was precipitated with ethanol, dried and resuspended in 0.01 M Tris-HCl, pH 7.5, 1 mM EDTA. 1 /~g of pSK DNA (native, DMS treated or uracil-containing) was mixed with 1/~g of pCM36 DNA and used for transformation according to the method of Mandel and Higa (1970). The transformation produced about 10 6 ampicillin resistance colonies per microgram of DNA. RecA protein isolation and assays of enzyme activity. RecA protein was isolated using the method of Cox et al. (1981). The ATPase activity of RecA protein was measured using the procedure of Shibata et al. (1979). Reaction mixtures contained 5-10 ~tmoles of RecA and 1 ttg of double-stranded plasmid DNA or 0.5 ~g of single-stranded bacteriophage M13 m p l 8 DNA. Results and discussion

To examine the mechanism of D N A recombination induced by mutagens and the role of D N A repair in this process, we have developed an in vivo plasmid recombination assay. The plasmids used are derived from pBR322 and contain nonoverlapping mutations in the tet gene (Fig. 1). Co-transformation of these plasmids into E. coli led to recombination between molecules Gf plasmid DNA and gave rise to tetracycline-resistant bacterial colonies. These colonies were counted and recombination frequencies were

TABLE 1 BACTERIAL STRAINS Strain

Relevant genetic markers

Source/Ref.

HB101

F , hsdS20 (rB-, roB- ), recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Smr), xyl-5, mtl-1, supE44, lambda as HB101, but recA ÷ Hfr KL16 PO/45 [cys (61-62)] dutl, ungl, thil, relA1, 2bd-279::Tn10, supE44 thr-1, leuB6, thi-1, lacY1, galK2, ara-14, xyl-5, m t l l , hisG4, argE3, tsx33, supE44, kdgK51, F-, rpsL31, del (gpt-proA) 62 as ABl157, but X::Tn5, tagA1, alkA1 as KL16 del (pncA-xth), but tagA1, ada

Boyer and RoulandDussoix, 1969 Bolivar et al., 1977 Kunkel, 1985

RR1 RZ1032 ABl157 GC4803 BK2012

B. Sedgwick B. Sedgwick B. Sedgwick

191 EcoR I H i n d l l l

EcoRI

SalG

~

TABLE 3

a~s

~

BamH I alGI

Fig. 1. Schematic representation of plasmids used in in vivo recombination system. Plasmid pCM36 contains a cluster of point mutations in the structural part of tet gene, shown as crosses (Salganik et ai., 1986), and plasmid pSK has a 15 nucleotides deletion in the promoter region of the same gene (Serpinski et al., 1982).

calculated as a ratio of tetracycline-resistant colonies to the total n u m b e r of transformants. R e c o m b i n a t i o n between plasmid molecules increased after D M S treatment in a dose-dependent m a n n e r (data not shown) and was dependent u p o n normal functioning of the recA gene of E. coil (Table 2). We have used two different substrate p S K plasmids in our recombination assay, one containing d a m a g e produced b y D M S and the other containing uracil in place of the correct base thymine. These were then co-transfected with und a m a g e d p C M 3 6 D N A . The data from 5 experiments are summarised in Table 2 and show that DMS-treated D N A is at least 20 times more efficient in stimulating recombination than uracilcontaining D N A . Both types of D N A lesions, p r o d u c e d by uracil incorporation (Lindahl et al., 1977) or D M S treament (Riazzudin and Lindahl, 1978; T h o m a s et al., 1982) are effectively repaired,

TABLE 2 FREQUENCIES OF PLASMID RECOMBINATION STIMULATED BY URACIL-CONTAINING OR DMSTREATED PLASMID DNA PlasmidDNA a

E. cofi strain

non treated uracil-containing DMS-treated

RRI (re.cA+ ) (6.3 + 1.2). 10- 5 (1.7 + 1.3). 10-4 (3.9 + 1.3)- 10- 3

HB101(recA-) < 10-6 < 10-6 (3.2 + 1.3)- 10 - 5

a DMS-treated pSK DNA was used for co-transformation with untreated pCM36 DNA.

RELATIVE STIMULATION OF PLASMID RECOMBINATION MEASURED ON REPAIR-DEFICIENT E. coli STRAINS E. coil

Relativegenotype

strain ABl157 GC4803 BK2012 a

wild-type as ABl157, but tagA1, alkA1 as ABl157, but tagA1, ada

Relative efficiency of stimulation a 17.7+4.6 11.7+4.4 10.3 + 3.9

Relative efficiency was calculated as a ratio of recombination frequency after co-transformatoin of DMS-treated pSK plasmid DNA with untreated pCM36 DNA to the frequency of recombinaton obtained after co-transformation of untreated plasmids.

but DMS-treated D N A is more efficient in stimulation of recombination than plasmid D N A containing uracil residues. One possible explanation of these results is that D M S stimulates recombination because one of the methylation adducts blocks D N A replication. However, this interpretation contradicts the observation that U V - i n d u c e d D N A damage effectively stimulates recombination between n o n replicating l a m b d a phage genomes (Smith and Hays, 1985). T o explain our results we propose that the main reason, or the first step, leading to enhancement of recombination after D M S treatment is not activation of D N A repair and appearance of repair intermediates (such as single-strand nicks or gaps) participating in the recombination process, but rather activation of recombination by increasing R e c A binding to mutagen-treated D N A . This conclusion is supported by the observation that mutations in E. coli genes responsible for repair of D N A d a m a g e d with methylating agents like D M S does not decrease significantly the level of stimulation of recombination by D M S (Table

3). Recently Lu et al. (Lu et al., 1986; Lu and Echols, 1987) have shown that R e c A protein, which catalyzes the formation of recombination complexes, binds more effectively to UV-treated duplex D N A than to untreated D N A . We propose that some changes in the secondary structure of D N A which are induced by D M S treatment, but not by misincorporation of uracil in place of

192

+

RecA + "O o~ N

o lo

+

+

60 5O 40

r30 n p<

20

UP-

o

o~

(Cunningham et al., 1980). In any case, repair processes are also involved in stimulation of recombination because even uracil incorporation produced a 3-fold increase of recombination level (Table 2). It is reasonable to speculate that repair processes can stimulate the second step of recombination by producing nicked molecules and thus activating strand exchange.

Acknowledgements

10 0

dsDNA

dsDNA

ssDNA

DMS treated dsDNA

A" U dsONA

Fig. 2. ATPase activity of RecA protein stimulated by uracilcontaining or DMS-treated duplex DNA. dsDNA, untreated double-stranded DNA; ssDNA, untreated single-stranded DNA; A : U dsDNA, uracil-containing double-stranded DNA; DMS dsDNA, DMS treated double-stranded DNA.

We t h a n k Dr. B. Sedgwick for E. coli strains GC4803 a n d BK2012, We are grateful to Drs. T. L i n d a h l a n d S. Keyse for helpful discussions. This work was completed in the Clare Hall L a b o r a t o ries of the Imperial C a n c e r Research F u n d a n d was s u p p o r t e d by a n I C R F travelling fellowship awarded to G.L.D.

References thymine, induce RecA p r o t e i n b i n d i n g a n d thus stimulate r e c o m b i n a t i o n . In order to verify our suggestion we have measured the s t i m u l a t i o n of A T P a s e activity of RecA p r o t e i n by both u r a c i l - c o n t a i n i n g or D M S - t r e a t e d duplex D N A (RecA b i n d i n g to the D N A molecules stimulates its A T P a s e activity, Shibata et al., 1979). The results of these experiments are shown in Fig. 2. The level of s t i m u l a t i o n of A T P a s e activity by uracil c o n t a i n i n g D N A was approximately the same as by native duplex D N A . In c o n t r a s t , D M S t r e a t m e n t of d u p l e x D N A d r a m a t i c a l l y increased the hydrolysis of A T P by RecA protein, a direct reflection of increased bin d i n g of RecA p r o t e i n to D M S - t r e a t e d D N A . It is reasonable to conclude from the results of the experiments presented above that uracil-cont a i n i n g D N A , which is actively repaired within the cells, is not so active in i n d u c t i o n of r e c o m b i n a tion events because RecA p r o t e i n does not recognize it as a d a m a g e d molecule. I n contrast, D M S t r e a t m e n t of D N A stimulates RecA b i n d i n g thus stimulating recombination. We believe that the increased b i n d i n g of RecA p r o t e i n to d a m a g e d duplex D N A m a y play a crucial role in the i n d u c t i o n of r e c o m b i n a t i o n by m u t a g e n s because RecA association with D N A is the rate-limiting step in the r e c o m b i n a t i o n process

Abbott, P.G. (1985a) Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in E. coli K12, Mol. Gen. Genet., 201, 129-132. Abbott, P.G. (1985b) Mutational and recombinational events in carcinogen-modifiedplasmid DNA: influence of host-cell repair genes, Mutation Res., 145, 25-34. Baker, R.M., and R.N. Haynes (1967) Ultraviolet-induced enhancement of recombination among lambda bacteriophages in ultraviolet-sensitive bacteria, Mol. Gen. Genet., 100, 166-177. Baker, R.M., and R.N. Haynes (1972) Ultraviolet-induced recombination and repair of parental lambda bacteriophages labeled by means of host-controlled modification, Virology, 50, 11-26. Bolivar, F., R.L. Rodriguez, P.J. Greene, M.C. Baltach, H.L. Heynecker, H.W. Boyer, J.H. Grossa and S. Falkow (1977) Construction and characterization of new cloning vehicles, II. A multipurpose cloning system, Gene, 2, 95-113. Boyer, H.M., and D. Rouland-Dussoix (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli, J. Mol. Biol., 41,459-473. Clewell, D.B., and D.R. Helinski (1969) Supercoiled DNAprotein complex in E. coli, Purification and induced conversion to an open circular form, Proc. Natl, Acad. Sci. (U.S.A.), 77, 7347-7351. Cox, M.M., K. McEntee and I.R. Lehman (1981) A simple and rapid procedure for the large scale purification of the RecA protein of E. coli, J. Biol. Chem., 265, 4676-4678. Cunningham, R.P., C. DasGupta and C.M. Radding (1980) Homologous pairing in genetic recombination: RecA protein makes joint molecules of gapped circular DNA and closed circular DNA, Cell, 20, 223-225.

193 Curtiss, R. (1968) Ultraviolet-induced genetic recombination in a partially diploid strain of E. coli, Genetics, 58, 9-54. Hays, J.B., B.K. Duncan and S. Boehmer (1981) Recombination of uracil-containing lambda bacteriophages, J. Bacteriol., 145, 306-320. Howard-Flanders, P., and R.P. Boyce (1966) DNA repair and genetic recombination: studies on mutants of E. coli defective in these processes, Radiat. Res., Suppl., 6, 165-184. Howard-Flanders, P., W.D. Rupp, B. Wilkins and R.S. Cole (1968) DNA replication and recombination after UVirradiation, Cold Spring Harbor Symp. Quant. Biol., 33, 195 - 207. Jacob, F., and E.U Wollman (1953) Induction of phage development in lysogenic bacteria, Cold Spring Harbor Symp. Quant. Biol., 18, 101-120. Lieb, M. (1985) Recombination in the lambda repressor gene: evidence that very short patch (VSP) mismatch correction restores a specific sequence, Mol. Gen. Genet., 199, 465470. Lindahl, T., S. Ljungquist, W. Seigert, B. Nyberg and B. Sperens (1977) Properties of uracil-DNA glycosylase from E. coli, J. Biol. Chem., 252, 3286-3294. Lu, C., and H.E. Echols (1987) RecA protein and SOS: correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage. J. Mol. Biol. 196, 497-504. Lu, C., R.H. Scheuermann and H. Echols (1986) Capacity of RecA protein to bind preferentially to UV lesions and inhibit editing subunit of DNA polymerase, IlL A possible mechanism for SOS-induced targeted mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 83, 619-623. Mandel, M., and A. Higa (1970) Calcium-dependent bacteriophage DNA infection, J. Mol. Biol. 53, 159-162. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular

Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Riazuddin, S., and T. Lindahl (1978) Properties of 3-methyladenine-DNA glycosylase from E. coli, Biochemistry, 17, 2110-2118. Sagher, D., and B. Strauss (1983) Insertion of nucleotides opposite apurinic/apirimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides, Biochemistry, 22, 4518-4526. Salganik, R.I., G.L. Dianov and O.A. Medvedev (1986) Cluster of point mutations predetermined by a quasipalindromic nucleotide sequence in plasmid pBR322 DNA, FEBS Lett., 261, 28-30. Serpinski, O.I., E.A. Karginova, N.N. Mikryukov et al. (1982) Constructon and properties of the vehicle for cloning the promoter-containing D N A fragments, The cloning of E. coli and phage T 7 promoters, Bioorganicheskaya Chimia (U.S.S.R.), 8, 840-847. Shibata, T., C. DasGupta, P. Cunningham and C.M. Radding (1979) Purified Escherichia coli RecA protein catalyzes homologous pairing of superhelical DNA and singlestranded fragments, Proc. Natl. Acad. Sci. (U.S.A.), 76, 1638-1642. Smith, A.G., and J.B. Hays (1985) Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli: stimulation of RecF dependent recombination by excision repair of cyclobutane pyrimidine dimers and other photoproducts, Mol. Gen. Genet., 201, 393-401. Taylor, W.D., C. Luisi-Deluca and R.D. Potter (1983) Carcinogen stimulated recombination in E. coli, J. Cell. Biochem., Suppl., 7B, 234-240. Thomas, U, C-H. Yang and D.A. Goldthwait (1982) Two DNA glycosylase in E. coli which release primary 3-methyladenine, Biochemistry, 21, 1162-1168.