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Methyl-Directed Repair of DNA Base-Pair Mismatches in Vitro

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Methyl-Directed Repair of DNA Base-Pair Mismatches in Vitro Proc. Natl. Acad. Sci. USA Vol. 80, pp. 4639-4643, August 1983 Biochemistry Methyl-directed repair of DNA base-pair mismatches in vitro (mutagenesis/gene conversion/DNA methylation) A.-LIEN Lu, SUSANNA CLARK, AND PAUL MODRICH Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Communicated by Robert L. Hill, April 18, 1983 ABSTRACT An assay has been developed that permits anal- system requires not only detection of base-pair mismatches but ysis of DNA mismatch repair in cell-free extracts of Escherichia a mechanism for discrimination of parental and newly synthe- coli The method relies on repair of heteroduplex molecules of fl sized strands as well. These authors suggested that the transient R229 DNA, which contain a base-pair mismatch within the single undermethylation of the newly synthesized strand might pro- EcoRI site of the molecule. As observed with mismatch hetero- vide the bias for such discrimination. Indeed, several lines of duplexes of A DNA [Pukila, P. J., Peterson, J., Herman, G., evidence indicate that dam methylation of d(G-A-T-C) se- Modrich, P. & Meselson, M. (1983) Genetics, in press], in vivo mis- quences functions in this respect. Thus, deficiency or over- match correction of fl heteroduplexes is directed by the state of production of this DNA methylase results in a mutator phe- dam methylation of d(G-A-T-C) sequences within the DNA du- notype (13, 14). In addition, genetic analysis has suggested that plex. Thus, the heteroduplex dam methylase participates in a pathway involving mutH, mutL, 5'-G-A-A-T-T-C and mutS function (15, 16). However, the most compelling evidence has been provided 3'-T-T-T-A-A-G by the transfection experiments of Meselson and colleagues (refs. is repaired in vivo to an EcoRI-sensitive form if the strand bearing 12 and 17; M. Rykowski and M. Meselson, personal commu- the wild-type EcoRI sequence carries the dam modification and nication), which employed A heteroduplexes in defined states the other does not. Such molecules are also subject to mismatch of dam methylation. Hemi-methylated heteroduplexes were repair by E. coli extracts. The in vitro activity is also dependent subject to mismatch repair almost exclusively on the unmeth- on ATP, the state of dam methylation of mismatch heterodu- ylated strand to yield the genotype of the methylated strand, plexes, and products of mutH, mutL, mutS, and uvrE loci. How- and repair was found to be substantially decreased if both strands ever, crude fractions deficient in these gene products do comple- of the heteroduplex were fully modified at d(G-A-T-C) se- ment in the cell-free system, thus providing assays for their isolation. quences. The in vitro reaction is accompanied by repair synthesis on the In this paper we describe a substrate that permits in vitro unmethylated DNA strand. analysis of mismatch repair. The reaction in crude cell fractions is dependent on the state of DNA methylation and requires Base-pair mismatches within the DNA double helix may arise ATP as well as the products of mutH, mutL, mutS, and uvrE. in several ways. Spontaneous deamination of cytosine or ad- enine leads to G-U or I-T mispairs, respectively. Such lesions MATERIALS AND METHODS are thought to be repaired via action of DNA glycosylases (1, 2). Mismatched base pairs may also occur during homologous Bacterial and Phage Strains. E. coli K38 (Hfr C; ref. 18) was genetic recombination if allelic differences are included within from R. E. Webster of this department; RS5033 (Hfr H, metBI the heteroduplex region formed by breakage and rejoining of rell strlO0 azi7 lacMS286 thi dam4 480dIIlacBKl), from E. B. parental molecules. The phenomenon of gene conversion has Konrad (19); andJC4583.(endAl ga144 thi-l thyA48 thyR27 lc6l), been attributed to repair of such mismatches (3). DNA repli- from A. J. Clark (University of-California, Berkeley). B. Glick- cation errors may also contribute to generation of mispaired man (National Institutes of Environmental Health Sciences) bases. In the case of the chromosome of Escherichia coli, the provided strains KMBL 3752 (endA101 thyA306 lysA65 argA103 error rate has been estimated to be 108 to 10-11 per base pair bio-87 metE72 pheA97 purA aroB cysC), KMBL 3773 (as KMBL replicated (4, 5), substantially lower than the in vitro error rate 3752 but mutHl01), KMBL 3774 (as KMBL 3752 but mutL101), of DNA replication systems derived from this organism (6). KMBL 3775 (as KMBL 3752 but mutS101), and KMBL 3789 (as Direct evidence for existence of a mechanism for correction KMBL 3752 but uvrE502) (15). of mismatched base pairs has been provided in the E. coli sys- Bacteriophage fl R229 containing an EcoRI site in the in- tem by transfection with A (7-9), 4X174 (10), and T7 hetero- tragenic region (20) was provided by R. E. Webster. Deriva- duplexes (11) marked genetically on the two DNA strands. Such tives containing mutations within the EcoRI sequence were experiments have demonstrated that incorrect base pairs can be constructed by minor modification of the procedure of Shortle eliminated from heteroduplexes prior to replication and, fur- and Nathans (21) and structure was determined by DNA se- thermore, have implicated the products of E. coli mutH, mutL, quence analysis according to Maxam and Gilbert (22). mutS, and uvrE loci in this process (9, 11, 12). Because strains DNA Preparations. Bacteriophage fl replicative form (RF) defective with respect to these loci exhibit a mutator phenotype I preparations were isolated (23) from chloramphenicol-treated (5), it seems likely that this set of genes directs a system in- infected cells (24). Single-stranded viral DNA was isolated from volved in postreplication repair of DNA biosynthetic errors. As purified virions (25, 26). DNAs devoid of dam methylation at pointed out by Wagner and Meselson (8), function of such a d(G-A-T-C) sequences were prepared by using strain RS5033 as host. DNAs modified at this sequence were prepared by us- The publication costs of this article were defrayed in part by page charge ing K38 or K38 harboring pGG503, a plasmid which leads to 10- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S.C. §1734 solely to indicate this fact. Abbreviations: RF, replicative form; kb, kilobase(s). 4639 Downloaded by guest on September 28, 2021 X%.rxo Biochemistry: Lu et al. Proc. Natl. Acad, Sci. USA 80 (1983) to 40-fold overproduction of the dam methylase (27). Table 1. Methyl-directed mismatch repair of fl R229 in vivo DNA heteroduplexes were prepared by mixing fl duplexes Transfectants (100 of RF III, linearized with HincII) with a 10-fold molar C/V ,ug Mixed Total excess of viral strands, followed by alkaline denaturation and methylation EcoRIP EcoRT/ annealing as described (28). After isolation by hydroxylapatite +/- 48 1 2 51 chromatography (29), double-stranded DNA was dialyzed against -/+ 0 17 0 17 0.01 M Tris-HCI, pH 8.0/1 mM EDTA, and then was subjected -/- 22 18 1 41 to closure with E. coli DNA ligase (30) in the presence of ethid- F1 heteroduplexes containing a G-T mismatch within the EcoRI site ium bromide (96 mmol of dye per mol of nucleotide). Cova- were constructed in several states of methylation. The state of dam lently closed DNA circles were then isolated by equilibrium methylation ofcomplementary (C) and viral (V) strands is indicated by centrifugation in CsCl/ethidium bromide as described (31). + and -, respectively. The strands are indicated as Hemi-methylated heteroduplexes prepared by using meth- C 5'-G-A-A-T-T-C ylated RF and unmethylated viral strands were resistant to cleavage by Mbo I, indicating that all d(G-A-T-C) sites were in V 3'-T-T-T-A-A-G. the hemi-methylated state. In contrast, hemi-methylated mol- In the case ofviral strand modification, incompletely hemi-methylated ecules prepared from unmethylated RF and methylated viral molecules were eliminated by Mbo I cleavage. Heteroduplexes were in- strands were subject to some cleavage by this enzyme. Because troduced into K38by transfection (35), and cells were immediately plated this problem was less severe if phage were propagated on the for infective centers. Individual plaques were picked into 1 ml of R broth overproducer of the dam methylase (above), this host was em- (32), which was then supplemented with 0.2 ml ofan overnight culture ployed for preparation of methylated viral strands. In this case, of K38. After incubation at 370C for 3-4 hr, a 0.1-ml sample was re- = 69% of the heteroduplexes were resistant to Mbo I, 21% were moved and used to infect a logarithmic culture of K38 (1.5 ml, A590 1.5 in Rbroth). Twenty minutes later chloramphenicol was added to 30 cleaved once, and 10% were cleaved more than once. If the tsg/ml, and after an additional 60 min cells were collected by centrif- unmethylated sites are distributed randomly with respect to ugation. RF DNAwas preparedby amini-lysate procedure (36) and tested the four d(G-A-T-C) sites of fl viral strands, a binomial distri- for cleavage byEcoRI endonuclease. DNA scored as sensitive to the en- bution with a probability of methylation of 0.9 for each d(G-A- zyme was cleaved to an extent of >95%, whereas resistant DNA was T-C) site would predict 66%, 29%, and 5% for these three classes. subject to <5% cleavage. Intermediate values were scored as mixed. Although perhaps not entirely random, it seems reasonable to conclude that methylation of viral single strands is about 90% ner et al.
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