Gene, 87 (1990) I-S Elsevier
GENE03~!
Specificity of Escherickia coil mutD and mutL mutator strains (Recombinant DNA; proofreading; mismatch repair; transition and transversion mutations; hot spots; plasmid pBR322; phage P22)
T.-H. Wu, C.H. Clarke and M.G. Marinus Department of Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655 (U.S.A.) Received by S.R. Kushner: 26 May 1989 Revised: 30 September 1989 Accepted: 31 September 1989
SUMMARY
The products of the mutD and mutL genes of Eschetchia coli are involved in proofreading by DNA polymerase I!1 and DNA adenine MTase (Dam)-dependent mismatch repair, respectively. We have used the plasmid-bome bacteriophage P22 rant gene as a target to determine the types of mutations produced in mut£25 and minD5 strains. Of 60 mutations identified from ntutL25 cells, 52 were transition mutations and ofthese the AT ~ GC subset predominated (40 out of 52). The majority of AT -* GC mutations were found at the same three sites (hotspots). In contrast, transversion mutations (4-/out of'/6) were found about twice as frequendy as transitions (28 out of-/6) from mutD5 bacteria. Two hotspots were identified but at diffcrent sites than those in the mutL25 cells. These results suggest that the proofreading function of DNA polymerase !ii primarily repairs potential transversion mutations while Dam-dependent mismatch repair rectifies potential transition mutations.
INTRODUCTION
There are at least two mechanisms by which mismatched bp are corrected in newly synthesized DNA: proofreading and post-replicative mismatch repair. Proofreading is intimately associated with replication and errors are removed as soon as they arise (Kornberg, 1980; 1988; Kunkel, 1988). The proofreading function is determined by the ~ and e sub-units of DNA polymerase II1 holoenzyme which are Correspondenceto: Dr. M.G. Marinus, Dept. of Pharmacology, University of Massachusetts Medical School, 55 Lake Ave., Worcester, MA 01655 (U.S.A.) Tel. (508)856-3330; Fax (508)856-$$15. Abbreviations: Ap, ampicillin; bla, gene encoding p-lactamase; bp, base pair(s); Dam, DNA adenine MTase; dam, gene encoding Dam; dis, heteroimmune (dismune); hy, hybrid; imm, immunity; IS, insertion sequence; mnt, bacteriophage P22 gene involved in maintenance of lysogeny; MTase, methyltransferase; rout, gene whose mutant allele results in a mutator phenotype; nt, nucleotide(s); O~, operator constitutive; or/, origin of DNA replication; R, resistance/resistant; s, sensitive; Tc, tetracycline; tet, gene in pBR322 encoding for TcR; tsp, transcription start point; wt, wild type. 0378-1119/90/$03.50 © 1990Elsevier Science Publishers B.V. (Biomedical Division)
specified by the polC and mutD (dnaQ)genes respectively (Scheuermann and Echols, 1984; DiFrancesco et ai., 1984; Cox and Homer, 1986; Maki and Komber8, 1987).Mutant polC and mutD strains of Escherfchla coil show an increased spontaneous mutation rate which is consistent with the postulated role of these gene products (Hail and Brammar, 1973; Degnan and Cox, 1974; Sevastopoulos and Glazer, 1977; Horiuchi et ai., 1978). The substrate for mismatch repair is duplex DNA containing the newly synthesized unmethylated daughter strand and a methylated parental strand (Claverys and Lacks, 1986; Radman and Wagner, 1986; Modrich, 1987; Meselson, 1988). Hemimethylated Dam-recognition sites (GATC) are substrates for incision by the MutH protein on the unmethylated strand. The MutS protein recognizes mismatched base pairs and the activities of the MutL, DNA helicase II and single-strand binding proteins are required for maximal repair (Modrich, 1987). Thus the bacteria mutants in the mutH, mutL, rams and uwD (DNA helicase II) genes show elevated spontaneous mutation rates (Cox, 19-/6).
The aim of the present studies is to show that the mutation spectrum is the same in mismatch repair-deficient or defective strains but different from that in proofreadingdefective bacteria. A preliminary account of this work was presented at the New England Biolabs Workshop on Biological DNA Modification (Carraway et al., 1988).
f o r w a r d m u t a t i o n s t h a t inactivate the rant gene or its o p e r a tor c o n f e r a T c a p h e n o t y p e . T h i s selective system h a s b e e n
MATERIALS AND METHODS
greater t h a n 95 % o f t h e T c a m u t a t i o n s m a p to the o p e r a t o r , b e t w e e n 5 0 - 9 0 % o f m e m u t a t i o n s from the m u t a t o r s t r a i n s
(a) Bacterial strains and plasmids Strains ES1582 (mutL25) and ES1578 (mutD5) are derivatives of AB1157 and were obtained from Dr. E. Siegel, Tufts University, Medford, MA. Plasmids F42 (F'lac) and pPY97 were introduced into these strains to yield GM3134 and GM3150, respectively. Strain MM294 was used as the host strain from which plasmid DNA was prepared for DNA sequencing and was obtained from the E. coli Genetic Stock Center (B. Bachmann, Curator, Dept. of Biology, Yale University, New Haven, CT). Plasmid pPY97 is a derivative of pBR322 containing the rant gene and an MI3 or/ (Lucchesi etal., 1986; Rewinski and Marinus, 1987).
are in t h e rant gene.
(b) Other methods Cells were grown in Brain Heart Infusion (BHI) broth at 37°C and bacteria and mutations in mnt were isolated as previously described (Lucchesi et ai,, 1986; Cart.way et al,, 1987; Rewinski and Marinus, 1987). Briefly, a portion from each of a series of overnight standing BHI broth cultures containing 40 ~g Ap/ml was plated on medium containing 3.5 #g Tc/ml and 40 ~g Ap/ml. Alter overnight incubation, colonies were restreaked twice on BHI broth plates containing 10 pg Tc/ml. The Tc R isolates were lysogenized with phage k/mme::hyl (Yamamoto et al., 1978) and challenged with phage ~[mmP22dts (Yamamoto et al., 1978). Lysogens sensitive to superinfection have mutations in the rant gene whereas those which are resistant contain mutations in the Mnt operator region (Lucchesi et al., 1986). Plasmids from Mnt- cells were transferred into strain MM294 and supercoiled plasmid DNA was isolated and sequenced as described by Rewinski and Marinus (1987).
RESULTS AND DISCUSSION
(a) Experimental system The rant gone of phage P22 was used as a target to monitor and identify the specificity of rout,genesis. This gone is present on a plasmid (pPY97) which was used to transform each mutator strain. Since the Tc a gone (tot) of pPY97 is under the negative control of the Mnt represser,
d e s c r i b e d in detail elsewhere ( L u c c h e s i e t a l . , 1986; C a r r a w a y et al., 1987; R e w i n s k i a n d M a r i n u s , 1987). S p o n t a n e o u s T c R cells arise in w t cultures at a f r e q u e n c y o f a b o u t I × 1 0 - s. F o r d a m - d e f i c i e n t strains, the f r e q u e n c y is i n c r e a s e d a b o u t 15-fold a n d for m u t H a n d m u t L m u t a n t s a b o u t 100-fold. T h e frequency in m u t D cultures is a b o u t 1000-fold a b o v e the w t level. I n c o n t r a s t to the wt, w h e r e
(b) Distribution and frequency of spontaneous mutations from the mutL25 strain Table I lists 60 independent mutations in the rant gone identified by n t sequencing w h i c h arose in the m u t L 2 5 TABLE 1 Analysis of mutations in the rant gone arising in a mutL2$ strain Position" (nt)
nt change
aa change
-44/-34 -11 24
Deletion A --. G G -. A
Promoter Promoter Met -. lie
A --. G C -, T A --, O C --, T G -, A
Arg2 --, Oly His6 -, Tyr His6 --, At8 Asns --, Ser Ar8 '° -, Cys Arg ' ° - . His
+C
FrameshlA
I
-G
Frameshilt Leu m .-, Ser Lys=° -, Ash Arg=a--, Met Frameshilt Alaa3 -, Val FrameshiA Glya~-. Trp Arga' -. Gly
I 2
28 4O 41 47 52 53 62-63 71-73 80
A --. O
T -* C
A -, C O --, T +.G 92 C --, T 103-105 ' ' " - G 103 ' • '.G --, T I06 A --, G
84 89 89-91
I09 If0 I15 116 121 122 149 273
T --. C C-. T A -* O
A --. G G --. A 'A ~ G
T -. C • A --. G
Ser=°-. Pro SeP~'-. Leu Met 3° - . Val
Asn3: --. Ser Glu 33-. Lys GIu 33 -, ely Leu4: ~-. Pro Stop -. Trp
Number"
I ! 2
I 2 16 I 2
I
I
I I 2 1 1 7 I 2
2 4 I 2
I 2
a Numbered with respect to the tsp ( + l)ofthe rant gene. The start codon
is at nt 21-23 and the stop codon at nt 271-273. Mutations were isolated and characterized from strain GM3134. a mutL2$ derivative of AB 1157 bearing plasmids F'42 a~d pPY97..as described in MATERIALS AND METHODS. The same methods were used to isolate the mutations described in Table III except that strain GM3150; a mute5 derivative of AB1157. was used. ' "". " b Number of identical mutants isolated. . •.
TABLE !I Type and frequency of mutations arising in the mm 8erie in wt and mutator strains Type of mutation
IS element Deletion Frameshi~ Transition AT ~ GC GC--, AT Transversion AT --. TA AT ~ CG GC-bCG GC ~ TA
Allele" (frequency of mutations b) vet
dam-3
mud134
mwL 2S
muzD5
10/13 3/! 3 0/13 0/! 3
6/90 2/90 5/90 73/90 62/73 ! 1/73 4/90 I/4 2/4 0/4 I/4
!/47 !/47 0/47 45/47 32/45 13/45 0/47
0/60 !/60 4/60 52/60 40/52 12/52 3/60 0/3 I/3 0/3 2/3
0/76 1/76 0/76 28/76 12/28 16/28 47/76 13/47 27/47 7/47 0/47
0113
" The data for the dam, mutH and wt strains are taken from Carraway et al. (1987) and Rewinski and Marinus (1987). The data for the other strains are from this paper (Tables I and II!). See footnotes to Table !. b The number of each type of mutation as a fraction of the total.
background. Table II summarizes the data for this strain as well as the wt and other mutator bacteria. In the mutL2$ cells, transition mutations comprised the major class (s2/6o) and ofthese the AT ~ GC subset predominated (4%2). The other eight identified mutations were a deletion, four frameshifts and three transversions, The deletion included nt -34 to -44 which eliminated the -35 region of the rant promoter, Three of the frameshifls involved insertion or deletion of a G residue in a run of three consecutive G (at nt 71-73, 89-91 and 103-105). Such frameshiAs are not unusual and are hypothesized to arise by 'slippage' of DNA strands (Streisinger et al., 1966). The fourth frameshift is unusual in that it is an insertion of a C residue between Tea
in Tables I and Ii which of these, or both, is responsible for the mutations we have detected. More recent data obtained by in vitro experiments indicate that both mispairs are formed for nmz (M. Carraway, C. Rewinski and M.G.M., unpublished data). The AT ~ GC class of mutations in ram, however, is three to six times more frequent than the GC ~ AT transition class. This bias is due to the frequent occurrence of AT -b GC transitions at the same three sites 0totspots), i.e., at nt positions 4!, 106 and 116. Fig. 1 shows that all three mismatch-repair-defective strains (dora-3, mutH34 and mutL25) yield these hotspots. The hotspot sites are within six bp of the only two Dam-recognition (GATC) sequences in the mnz gene, at nt positions 34-37 and 107-110. Table II shows that no transition or transversion mutations were detected in the wt strain. Systems which repair mismatches, therefore, must be very efficient in the wt background.
.°1,.°u, ,o2,11 T-°°,,. ,~
'
(n
,o
mutH34 AT -~ GC
t| I
•
a n d G 63.
E (e) Comparison of mutation spectra in strains defective in mismatch repair Table ll compares the mutation spectra in dam-3 (Carraway et al., 1987), mutH34 (Rewinski and Marinus, 1987) and mutL25 strains. They are strikingly similar to each other, but are different to those of the wt and mutD5 strains. In particular, the strains defective in mismatch repair yielded primarily transition mutations and, at a lower frequency, transversions and frameshifts. In these respects, the results with rant are in agreement with those obtained in the lacl system (Leong et al., 1986; Schaaper and Dunn, 1987). The combined data argue that the products of all these genes are involved in the rectification of nt mispairs by Dam-dependent mismatch repair. The transition mutations can arise from either A. C or G. T mispairs. It is not possible to determine from the data
,'.
n
n
,'oo
,]
'01 it t
.
.o
il •
NL ii TeA
nt Number Fig. I. Location of AT--, GC mutational hotspots in the mm 8ene obtained from the indicated bacterial strains. The number of AT-, GC transitions at selectednucleotides in the mm ~ne from mutL2$ (Table !), dam-3 and mutH34 strains (Can'away et al., 1987; Rewinski and Marines (1987) is displayed. The three hotspots at nt positions 41~ 106 and 116 are present in all these strains. They were not prominent in either the mutD$ (Table !I!)or the wt strains (Can'away et 81., 1987; Rewinsky and Marinus, 1987). Numbering ofnt begins at the up (Lucehui et al., 1986). The mutations at nt 273 are in the stop qaxlon.
(d) Distribution and frequency of spontaneous mutations from the mutD5 strain Table III lists 76 mutations in rant which we~e recovered from mutD5 cells. These represent 60% of the total Tc R isolates; the remainder were phenotypically O c, L,xcontrast to the other mutator strains (Table II), transversion mutations were about twice as frequent (47/76) as transitions (28/v6), Apart from the two hotspot sites described below, the mutations were uniformly distributed throughout the proximal part of the rant gene and in the translational termination codon. Table lIl also shows that a deletion mutation was recovered which eliminated bp 194-199. This was the only deletion mutation greater than one bp that we have ever recovered in the rant coding region. The distribution of transition mutations from mutD5 bacteria was distinctly different from those of the other mutator strains (Table II). The hotspots characteristic of mismatchrepair-defective strahss at nt 41, 106 and 116 were absent and a new hotspot site was located at nt -33 in the promoter region (Table III). Also in contrast to the mutator strains described in Table II, the number of A T ~ GC
TABLE III Mutations in the mnt gene arising in a mutD5 strain Position ~'
nt change
aa change
Number b
-36 -35 -33 24 26 28 40 41 41 42 46 53 55 70 77 91 103 106 108 109 109 110 116 137 176 194-199 199
A -, C T~ C G -, A G -~ A C -* T A~ G C~ T A --*T A --* G C ,...*G A -~ T G --, A A~ T A -* T A -~ C G~ C G~ A A -, G A -* T T -~ G T -, A C -~ T A~ G T~ G A --, C Deletion G --* C
Promoter Promoter Promoter fMet ~ lie Ala* ~ Val Arg 2 -~ Gly His 6 --*Tyr His 6--, Leu His c' -* Arg His 6 --, Gin Asn s ~ lie Arg m -, His Met *i ~ Leu Arg te --, Trp Lys *~~ Thr Ala 2~ ~ Pro Gly 2v ~ Arg Arg 2s ~ Gly Arg 2s ~ Stop Ser 29 ~ Ala Ser 29 -~ Thr Ser 29 -, Leu Asn ~l -~ Ser VaP s ~ Gly Tyr sl ~ Ser Ala s9 ~ Pro
3 3 9 I I I 2 2 i 3 ! 1 I 3 2 3 ! 1 i 20 5 1 2 1 1 1 I
273
A --. G
Stop -, Trp
4
,,.b See footnotes a. b to Table I.
changes is approximately equal to the GC--, AT class in mutD5 cells. The majority ofthe transversions occurred at A. T bp (40 out of 47) and of these 25 changed T m9 to A or G (Table III). This T residu~ is vdthh~ the. Da,'~.-rc~c~i~,~,~n ' ~ site at nt 107-110. Since no hotspot was detected at the other GATC in rant, the mechanise~,of hotspot f o r m a ~ : must be independent of Dam-recognition sites. The data in Table III indicate that under the exf~rimental conditions we have used to isolate mutations in rant there is little, if any, inhibition of Dam-dependent repair in mutD5 bacteria since the spectrum of transition mutations does not resemble that for mismatch repair defective strains (cf., Schaaper, 1988; Isbell and Fowler, 1989). Consequently, it is reasonable to conclude that in the absence of proper proofreading by DNA polymerase III in mutD5 cells both transition and transversion mutations ar,.~ produced in rant with the latter comprising the major class. (e) Intergenic deletions were not included in the analysis For each of the mutator strains listed in Table II, about 1-10% ofthe Tc R isolates were Ap s. This is due to deletion mutations which have one end at variable sites within rant and the other end at variable points in the gene (bla) encoding ~-Iactamase. We have not included these deletions in the analysis because this kind of deletion was found in pBR322 derivatives without the rant gene (Yi et ai., 1988; S. Bouvier and M.G.M., unpublished data). We believe, therefore, that these deletions are a reflection of the plasmid nature of the target and may not be representative of events occurring on the host chromosome.
(f) An unexpected feature An unexpected feature of the spectra in Tables I and III, is that mutations are found almost exclusively in the DNA corresponding to the N-terminal half of the protein. At present, it is unclear why this should be. The Mnt repressor does not display the helix-turn-helix motif found in many DNA-binding proteins (Knight et al., 1989) and residues in both N-terminal (Knight and Sauer, 1989) and C-terminal (Knight and Sauer, 1988) parts of the repressor are necessary for DNA binding. Mnt repressor binds to DNA as a tetramer (Vershon et al., 1985) and to both the 'front' and 'back' of the operator (Vershon et al., 1987). Given these properties one might expect that frameshift or nonsense mutations affecting the C-terminal end of the repressor should be recovered in vivo from the mutator swains. That they have not been found in vivo is puzzling, especially since nonsense mutations affecting the C-terminal residues which perturb DNA binding have been isolated by site-directed mutagenesis (Knight and Sauer, 1988).
ACKNOWLEDGEMENTS W e t h a n k Dr. Eli Siegel for the gi~ o f bacterial strains. This w o r k w a s s u p p o r t e d by grant G M 3 3 2 3 3 from the N a t i o n a l Institutes o f Health.
REFERENCES Carraway, M., Youderian, P. and Marinus, M.G.: Spontaneous mutations occur near Dam recognition sites in a dam- Escherichia coli host. Genetics 116 (1987) 343-347. Carraway, M., Rewinski, C, Wu, T.-H. and Marinus, M.G.: Specificity ofthe Dam-directed mismatch repair system of Escherichia coil K-12. Gene 74 (1988) 157-158. Claverys, J.P. and Lacks, S.A.: l-leteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiol. Rev. 50 (1986) 133-165. Cox, E.C.: Bacterial mutator genes and the control of spontaneous mutation. A'nnu. Rev. Genet. 10 (1976) 135-156. Cox, E.C. and Homer, D.L.: DNA sequence and coding properties of mutD(dnaQ), a dominant E. coil mutator 8ene. J. Mol. Biol. 190 (1986) !13-117. Degnan, G.E. and Cox, E.C.: Conditional mutator gene in Escherichia coil: isolation, mapping and effector studies. J. Bacteriol. 177 (1974) 477-487. DiFrancesco, R.S., Bhatnagar, S.K., Brown, A. and Bessman, MJ.: The interaction ofDNA polymerase ill and the product ofthe Escherichia coli mutator gene, mull). J. Biol. Chem. 259 (1984) 5567-5573. Hall, R.M. and Brammar, WJ.: Increased spontaneous mutation rates in mutants of E. coil with altered DNA polymerase I11. Mol. Gen. Genet. 121 (1973)271-276. Horiuchi, T., Maki, H. and Sekiguchi, M.: A new conditional lethal mutator (dna(249) in Escherichla coil KI2. Mol. Gen. Genet. 163 (1978) 277-283. lsbell, R.J. and Fowler, R.G.: Temperature-dependent mutational specificity of an £sche~chla colt mutator, dnaO.49, defective in 3' to $' exonuclease (proofreading) activity. Murat. Res. 213 (1989) 149-156. Knight, K,L. and Sauer, R.T.: The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation. Biochemistry 27 (1988) 2088-2094. Knight, K.L. and Sauer, R.T.: DNA binding specificity of the Arc and Mat repressors is determined by a short region of N-terminal residues. Proc. Natl. Acad. Sci. USA 86 (1989) 797-801. Knight, K.L., Bowie, J.U., Vershon, A.K., Kelley, R.D. and Saner, R.T.: The Arc and Mat repressors. J. Biol. Chem. 264 (1989) 3639-3642. Kornberg, A.: DNA Replication. Freeman and Co., San Francisco, CA, 1980. Kornberg, A.: DNA replication. J. Biol. Chem. 263 (1988) I-4.
Kunkel, T.A.: Exonucleolytic proofreading. Cell 53 (1988) 837-840. Leong, P-M., Hsi8, H.C. and Miller, J.H.: Analysis of spontaneous base substitutions generated in mismatch-repair deficient strains of EscAe~/a cob: J. Bacteriol. 168 (1986) 412-416. Lucchesi, P., Can'away, M. and Marinus, M.G.: Avalysis of forward mutations induced by N.methyl.N-rfitro-N.rdtros~uam~dlne in the bacteriophage P22 mm repressor gene. J. Bacteriol. 166 (1986) 34-37. Maki, H. and Komberg, A.: Proofreading by DNA polymerase 111 of" Es~erichia ¢oUdepends on cooperative interaction of the polymerase and exonuclease subunits. Proc. Natl. AcacL Sci. USA 84 (1987) 4389--4392. Meselson, M.: Methyl-directed repair of DNA mismatches. In Low, K.B. (Ed.), The Recombination of Genetic Material. Academic Press, New York, 1988, pp. 91-113. Modrich, P.: DNA mismatch correction. Annu. Revo Biochem. 56 (1987) 435-466. Radman, M. and Wagner, R.: Mismatch repair in Escherichia coti. Annu. Rev. Genet. 20 (1986) 523-538. Rewinski, C. and Marinus, M.G.: Mutation spectrum in an F.s~erich~ coil DNA mismatch repair deficient (mutH) strain. Nucleic Acids Res. 15 (1987) 8205-8215. Schaaper, R.M.: Mechanisms ofmutagenesis in the E s c h e t c ~ cob"mutamr mutDS: role of DNA mismatch repair. Proc. Natl. Acad. Sci. USA 85 (1988) 8126-8130. Schaaper, R.M. and Dunn, R.L.: Spectra of spontaneous mutations in Escherichia coil strains defective in mismatch correction: the nature of in vivo replication errors. Proc. Natl. Acad. Sci. USA 84 (1987) 6220-6224. Scheuermann, R.H. and Echols, H.: A separate editing exonuclease for DNA replication: the epsilon subunit of£scherichia coil DNA polymerase I!! holoenzyme. Proc. Natl. Acad. Sci. USA 81 (1984) 7747-7751. Sevastopoulos, C.G. and Glaser, D.A.: Mutator action by Escherichia coli strains carrying dnaE mutations. Proc. Natl. Acad. Sci. USA 74 (1977) 3947-3950. Streisinaer, G., lkada, Y., Emrich, J., Newton, J., Tsuaita, A., Terzhaai, E. an d lnouye, M.: Frameshit~ mutations and the genetic code. Cold Spring Harbor Syrup. Quant. Biol. 31 (1966) 77-84. Vershon, A.K., Youderian, P., Susskind, M.M. and Saner, R.T.: The bacteriophage P22 Arc and Mat repressors: overproduction, purifi. cation and properties. J. Biol. Chem. 260 (1985) 12124-12129. Vershon, A.K., Liao, S,-M., McClure, W.R. and Sauer, R.T.: Bacteriophage P22 Mat repressor. DNA bindin8 and effects on transcription in vitro. J. Mol. Biol. 195 (1987) 311-322. Yamamoto, N., Wohlheiter, J.A., Gemski, P. and Baron, L.S.: ,]/mmP22d/s: a hybrid of coliphage ,~ with both immunity regions of Salmonella phage P22. Mol. Gen. Genet. 166 (1978) 233-243. Yi, T.-M., Stearns, D. and Demple, B.: illegitimate recombination in an Escherichia coil plasmid: modulation by DNA damage and a new bacterial gene. J. Bacteriol. 170 (1988) 2898-2903.
GENE03~!
Specificity of Escherickia coil mutD and mutL mutator strains (Recombinant DNA; proofreading; mismatch repair; transition and transversion mutations; hot spots; plasmid pBR322; phage P22)
T.-H. Wu, C.H. Clarke and M.G. Marinus Department of Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655 (U.S.A.) Received by S.R. Kushner: 26 May 1989 Revised: 30 September 1989 Accepted: 31 September 1989
SUMMARY
The products of the mutD and mutL genes of Eschetchia coli are involved in proofreading by DNA polymerase I!1 and DNA adenine MTase (Dam)-dependent mismatch repair, respectively. We have used the plasmid-bome bacteriophage P22 rant gene as a target to determine the types of mutations produced in mut£25 and minD5 strains. Of 60 mutations identified from ntutL25 cells, 52 were transition mutations and ofthese the AT ~ GC subset predominated (40 out of 52). The majority of AT -* GC mutations were found at the same three sites (hotspots). In contrast, transversion mutations (4-/out of'/6) were found about twice as frequendy as transitions (28 out of-/6) from mutD5 bacteria. Two hotspots were identified but at diffcrent sites than those in the mutL25 cells. These results suggest that the proofreading function of DNA polymerase !ii primarily repairs potential transversion mutations while Dam-dependent mismatch repair rectifies potential transition mutations.
INTRODUCTION
There are at least two mechanisms by which mismatched bp are corrected in newly synthesized DNA: proofreading and post-replicative mismatch repair. Proofreading is intimately associated with replication and errors are removed as soon as they arise (Kornberg, 1980; 1988; Kunkel, 1988). The proofreading function is determined by the ~ and e sub-units of DNA polymerase II1 holoenzyme which are Correspondenceto: Dr. M.G. Marinus, Dept. of Pharmacology, University of Massachusetts Medical School, 55 Lake Ave., Worcester, MA 01655 (U.S.A.) Tel. (508)856-3330; Fax (508)856-$$15. Abbreviations: Ap, ampicillin; bla, gene encoding p-lactamase; bp, base pair(s); Dam, DNA adenine MTase; dam, gene encoding Dam; dis, heteroimmune (dismune); hy, hybrid; imm, immunity; IS, insertion sequence; mnt, bacteriophage P22 gene involved in maintenance of lysogeny; MTase, methyltransferase; rout, gene whose mutant allele results in a mutator phenotype; nt, nucleotide(s); O~, operator constitutive; or/, origin of DNA replication; R, resistance/resistant; s, sensitive; Tc, tetracycline; tet, gene in pBR322 encoding for TcR; tsp, transcription start point; wt, wild type. 0378-1119/90/$03.50 © 1990Elsevier Science Publishers B.V. (Biomedical Division)
specified by the polC and mutD (dnaQ)genes respectively (Scheuermann and Echols, 1984; DiFrancesco et ai., 1984; Cox and Homer, 1986; Maki and Komber8, 1987).Mutant polC and mutD strains of Escherfchla coil show an increased spontaneous mutation rate which is consistent with the postulated role of these gene products (Hail and Brammar, 1973; Degnan and Cox, 1974; Sevastopoulos and Glazer, 1977; Horiuchi et ai., 1978). The substrate for mismatch repair is duplex DNA containing the newly synthesized unmethylated daughter strand and a methylated parental strand (Claverys and Lacks, 1986; Radman and Wagner, 1986; Modrich, 1987; Meselson, 1988). Hemimethylated Dam-recognition sites (GATC) are substrates for incision by the MutH protein on the unmethylated strand. The MutS protein recognizes mismatched base pairs and the activities of the MutL, DNA helicase II and single-strand binding proteins are required for maximal repair (Modrich, 1987). Thus the bacteria mutants in the mutH, mutL, rams and uwD (DNA helicase II) genes show elevated spontaneous mutation rates (Cox, 19-/6).
The aim of the present studies is to show that the mutation spectrum is the same in mismatch repair-deficient or defective strains but different from that in proofreadingdefective bacteria. A preliminary account of this work was presented at the New England Biolabs Workshop on Biological DNA Modification (Carraway et al., 1988).
f o r w a r d m u t a t i o n s t h a t inactivate the rant gene or its o p e r a tor c o n f e r a T c a p h e n o t y p e . T h i s selective system h a s b e e n
MATERIALS AND METHODS
greater t h a n 95 % o f t h e T c a m u t a t i o n s m a p to the o p e r a t o r , b e t w e e n 5 0 - 9 0 % o f m e m u t a t i o n s from the m u t a t o r s t r a i n s
(a) Bacterial strains and plasmids Strains ES1582 (mutL25) and ES1578 (mutD5) are derivatives of AB1157 and were obtained from Dr. E. Siegel, Tufts University, Medford, MA. Plasmids F42 (F'lac) and pPY97 were introduced into these strains to yield GM3134 and GM3150, respectively. Strain MM294 was used as the host strain from which plasmid DNA was prepared for DNA sequencing and was obtained from the E. coli Genetic Stock Center (B. Bachmann, Curator, Dept. of Biology, Yale University, New Haven, CT). Plasmid pPY97 is a derivative of pBR322 containing the rant gene and an MI3 or/ (Lucchesi etal., 1986; Rewinski and Marinus, 1987).
are in t h e rant gene.
(b) Other methods Cells were grown in Brain Heart Infusion (BHI) broth at 37°C and bacteria and mutations in mnt were isolated as previously described (Lucchesi et ai,, 1986; Cart.way et al,, 1987; Rewinski and Marinus, 1987). Briefly, a portion from each of a series of overnight standing BHI broth cultures containing 40 ~g Ap/ml was plated on medium containing 3.5 #g Tc/ml and 40 ~g Ap/ml. Alter overnight incubation, colonies were restreaked twice on BHI broth plates containing 10 pg Tc/ml. The Tc R isolates were lysogenized with phage k/mme::hyl (Yamamoto et al., 1978) and challenged with phage ~[mmP22dts (Yamamoto et al., 1978). Lysogens sensitive to superinfection have mutations in the rant gene whereas those which are resistant contain mutations in the Mnt operator region (Lucchesi et al., 1986). Plasmids from Mnt- cells were transferred into strain MM294 and supercoiled plasmid DNA was isolated and sequenced as described by Rewinski and Marinus (1987).
RESULTS AND DISCUSSION
(a) Experimental system The rant gone of phage P22 was used as a target to monitor and identify the specificity of rout,genesis. This gone is present on a plasmid (pPY97) which was used to transform each mutator strain. Since the Tc a gone (tot) of pPY97 is under the negative control of the Mnt represser,
d e s c r i b e d in detail elsewhere ( L u c c h e s i e t a l . , 1986; C a r r a w a y et al., 1987; R e w i n s k i a n d M a r i n u s , 1987). S p o n t a n e o u s T c R cells arise in w t cultures at a f r e q u e n c y o f a b o u t I × 1 0 - s. F o r d a m - d e f i c i e n t strains, the f r e q u e n c y is i n c r e a s e d a b o u t 15-fold a n d for m u t H a n d m u t L m u t a n t s a b o u t 100-fold. T h e frequency in m u t D cultures is a b o u t 1000-fold a b o v e the w t level. I n c o n t r a s t to the wt, w h e r e
(b) Distribution and frequency of spontaneous mutations from the mutL25 strain Table I lists 60 independent mutations in the rant gone identified by n t sequencing w h i c h arose in the m u t L 2 5 TABLE 1 Analysis of mutations in the rant gone arising in a mutL2$ strain Position" (nt)
nt change
aa change
-44/-34 -11 24
Deletion A --. G G -. A
Promoter Promoter Met -. lie
A --. G C -, T A --, O C --, T G -, A
Arg2 --, Oly His6 -, Tyr His6 --, At8 Asns --, Ser Ar8 '° -, Cys Arg ' ° - . His
+C
FrameshlA
I
-G
Frameshilt Leu m .-, Ser Lys=° -, Ash Arg=a--, Met Frameshilt Alaa3 -, Val FrameshiA Glya~-. Trp Arga' -. Gly
I 2
28 4O 41 47 52 53 62-63 71-73 80
A --. O
T -* C
A -, C O --, T +.G 92 C --, T 103-105 ' ' " - G 103 ' • '.G --, T I06 A --, G
84 89 89-91
I09 If0 I15 116 121 122 149 273
T --. C C-. T A -* O
A --. G G --. A 'A ~ G
T -. C • A --. G
Ser=°-. Pro SeP~'-. Leu Met 3° - . Val
Asn3: --. Ser Glu 33-. Lys GIu 33 -, ely Leu4: ~-. Pro Stop -. Trp
Number"
I ! 2
I 2 16 I 2
I
I
I I 2 1 1 7 I 2
2 4 I 2
I 2
a Numbered with respect to the tsp ( + l)ofthe rant gene. The start codon
is at nt 21-23 and the stop codon at nt 271-273. Mutations were isolated and characterized from strain GM3134. a mutL2$ derivative of AB 1157 bearing plasmids F'42 a~d pPY97..as described in MATERIALS AND METHODS. The same methods were used to isolate the mutations described in Table III except that strain GM3150; a mute5 derivative of AB1157. was used. ' "". " b Number of identical mutants isolated. . •.
TABLE !I Type and frequency of mutations arising in the mm 8erie in wt and mutator strains Type of mutation
IS element Deletion Frameshi~ Transition AT ~ GC GC--, AT Transversion AT --. TA AT ~ CG GC-bCG GC ~ TA
Allele" (frequency of mutations b) vet
dam-3
mud134
mwL 2S
muzD5
10/13 3/! 3 0/13 0/! 3
6/90 2/90 5/90 73/90 62/73 ! 1/73 4/90 I/4 2/4 0/4 I/4
!/47 !/47 0/47 45/47 32/45 13/45 0/47
0/60 !/60 4/60 52/60 40/52 12/52 3/60 0/3 I/3 0/3 2/3
0/76 1/76 0/76 28/76 12/28 16/28 47/76 13/47 27/47 7/47 0/47
0113
" The data for the dam, mutH and wt strains are taken from Carraway et al. (1987) and Rewinski and Marinus (1987). The data for the other strains are from this paper (Tables I and II!). See footnotes to Table !. b The number of each type of mutation as a fraction of the total.
background. Table II summarizes the data for this strain as well as the wt and other mutator bacteria. In the mutL2$ cells, transition mutations comprised the major class (s2/6o) and ofthese the AT ~ GC subset predominated (4%2). The other eight identified mutations were a deletion, four frameshifts and three transversions, The deletion included nt -34 to -44 which eliminated the -35 region of the rant promoter, Three of the frameshifls involved insertion or deletion of a G residue in a run of three consecutive G (at nt 71-73, 89-91 and 103-105). Such frameshiAs are not unusual and are hypothesized to arise by 'slippage' of DNA strands (Streisinger et al., 1966). The fourth frameshift is unusual in that it is an insertion of a C residue between Tea
in Tables I and Ii which of these, or both, is responsible for the mutations we have detected. More recent data obtained by in vitro experiments indicate that both mispairs are formed for nmz (M. Carraway, C. Rewinski and M.G.M., unpublished data). The AT ~ GC class of mutations in ram, however, is three to six times more frequent than the GC ~ AT transition class. This bias is due to the frequent occurrence of AT -b GC transitions at the same three sites 0totspots), i.e., at nt positions 4!, 106 and 116. Fig. 1 shows that all three mismatch-repair-defective strains (dora-3, mutH34 and mutL25) yield these hotspots. The hotspot sites are within six bp of the only two Dam-recognition (GATC) sequences in the mnz gene, at nt positions 34-37 and 107-110. Table II shows that no transition or transversion mutations were detected in the wt strain. Systems which repair mismatches, therefore, must be very efficient in the wt background.
.°1,.°u, ,o2,11 T-°°,,. ,~
'
(n
,o
mutH34 AT -~ GC
t| I
•
a n d G 63.
E (e) Comparison of mutation spectra in strains defective in mismatch repair Table ll compares the mutation spectra in dam-3 (Carraway et al., 1987), mutH34 (Rewinski and Marinus, 1987) and mutL25 strains. They are strikingly similar to each other, but are different to those of the wt and mutD5 strains. In particular, the strains defective in mismatch repair yielded primarily transition mutations and, at a lower frequency, transversions and frameshifts. In these respects, the results with rant are in agreement with those obtained in the lacl system (Leong et al., 1986; Schaaper and Dunn, 1987). The combined data argue that the products of all these genes are involved in the rectification of nt mispairs by Dam-dependent mismatch repair. The transition mutations can arise from either A. C or G. T mispairs. It is not possible to determine from the data
,'.
n
n
,'oo
,]
'01 it t
.
.o
il •
NL ii TeA
nt Number Fig. I. Location of AT--, GC mutational hotspots in the mm 8ene obtained from the indicated bacterial strains. The number of AT-, GC transitions at selectednucleotides in the mm ~ne from mutL2$ (Table !), dam-3 and mutH34 strains (Can'away et al., 1987; Rewinski and Marines (1987) is displayed. The three hotspots at nt positions 41~ 106 and 116 are present in all these strains. They were not prominent in either the mutD$ (Table !I!)or the wt strains (Can'away et 81., 1987; Rewinsky and Marinus, 1987). Numbering ofnt begins at the up (Lucehui et al., 1986). The mutations at nt 273 are in the stop qaxlon.
(d) Distribution and frequency of spontaneous mutations from the mutD5 strain Table III lists 76 mutations in rant which we~e recovered from mutD5 cells. These represent 60% of the total Tc R isolates; the remainder were phenotypically O c, L,xcontrast to the other mutator strains (Table II), transversion mutations were about twice as frequent (47/76) as transitions (28/v6), Apart from the two hotspot sites described below, the mutations were uniformly distributed throughout the proximal part of the rant gene and in the translational termination codon. Table lIl also shows that a deletion mutation was recovered which eliminated bp 194-199. This was the only deletion mutation greater than one bp that we have ever recovered in the rant coding region. The distribution of transition mutations from mutD5 bacteria was distinctly different from those of the other mutator strains (Table II). The hotspots characteristic of mismatchrepair-defective strahss at nt 41, 106 and 116 were absent and a new hotspot site was located at nt -33 in the promoter region (Table III). Also in contrast to the mutator strains described in Table II, the number of A T ~ GC
TABLE III Mutations in the mnt gene arising in a mutD5 strain Position ~'
nt change
aa change
Number b
-36 -35 -33 24 26 28 40 41 41 42 46 53 55 70 77 91 103 106 108 109 109 110 116 137 176 194-199 199
A -, C T~ C G -, A G -~ A C -* T A~ G C~ T A --*T A --* G C ,...*G A -~ T G --, A A~ T A -* T A -~ C G~ C G~ A A -, G A -* T T -~ G T -, A C -~ T A~ G T~ G A --, C Deletion G --* C
Promoter Promoter Promoter fMet ~ lie Ala* ~ Val Arg 2 -~ Gly His 6 --*Tyr His 6--, Leu His c' -* Arg His 6 --, Gin Asn s ~ lie Arg m -, His Met *i ~ Leu Arg te --, Trp Lys *~~ Thr Ala 2~ ~ Pro Gly 2v ~ Arg Arg 2s ~ Gly Arg 2s ~ Stop Ser 29 ~ Ala Ser 29 -~ Thr Ser 29 -, Leu Asn ~l -~ Ser VaP s ~ Gly Tyr sl ~ Ser Ala s9 ~ Pro
3 3 9 I I I 2 2 i 3 ! 1 I 3 2 3 ! 1 i 20 5 1 2 1 1 1 I
273
A --. G
Stop -, Trp
4
,,.b See footnotes a. b to Table I.
changes is approximately equal to the GC--, AT class in mutD5 cells. The majority ofthe transversions occurred at A. T bp (40 out of 47) and of these 25 changed T m9 to A or G (Table III). This T residu~ is vdthh~ the. Da,'~.-rc~c~i~,~,~n ' ~ site at nt 107-110. Since no hotspot was detected at the other GATC in rant, the mechanise~,of hotspot f o r m a ~ : must be independent of Dam-recognition sites. The data in Table III indicate that under the exf~rimental conditions we have used to isolate mutations in rant there is little, if any, inhibition of Dam-dependent repair in mutD5 bacteria since the spectrum of transition mutations does not resemble that for mismatch repair defective strains (cf., Schaaper, 1988; Isbell and Fowler, 1989). Consequently, it is reasonable to conclude that in the absence of proper proofreading by DNA polymerase III in mutD5 cells both transition and transversion mutations ar,.~ produced in rant with the latter comprising the major class. (e) Intergenic deletions were not included in the analysis For each of the mutator strains listed in Table II, about 1-10% ofthe Tc R isolates were Ap s. This is due to deletion mutations which have one end at variable sites within rant and the other end at variable points in the gene (bla) encoding ~-Iactamase. We have not included these deletions in the analysis because this kind of deletion was found in pBR322 derivatives without the rant gene (Yi et ai., 1988; S. Bouvier and M.G.M., unpublished data). We believe, therefore, that these deletions are a reflection of the plasmid nature of the target and may not be representative of events occurring on the host chromosome.
(f) An unexpected feature An unexpected feature of the spectra in Tables I and III, is that mutations are found almost exclusively in the DNA corresponding to the N-terminal half of the protein. At present, it is unclear why this should be. The Mnt repressor does not display the helix-turn-helix motif found in many DNA-binding proteins (Knight et al., 1989) and residues in both N-terminal (Knight and Sauer, 1989) and C-terminal (Knight and Sauer, 1988) parts of the repressor are necessary for DNA binding. Mnt repressor binds to DNA as a tetramer (Vershon et al., 1985) and to both the 'front' and 'back' of the operator (Vershon et al., 1987). Given these properties one might expect that frameshift or nonsense mutations affecting the C-terminal end of the repressor should be recovered in vivo from the mutator swains. That they have not been found in vivo is puzzling, especially since nonsense mutations affecting the C-terminal residues which perturb DNA binding have been isolated by site-directed mutagenesis (Knight and Sauer, 1988).
ACKNOWLEDGEMENTS W e t h a n k Dr. Eli Siegel for the gi~ o f bacterial strains. This w o r k w a s s u p p o r t e d by grant G M 3 3 2 3 3 from the N a t i o n a l Institutes o f Health.
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