Evidence Suggesting a Mismatch Repair Mechanism

In standard crosses, some rIIB mutants of T4 phage were found to be susceptible to an extra recombination mechanism to which the other mutants were much less susceptible. The following observations were interpreted as evidence for the mismatch-repair nature of the phenomenon: (1) Marker-dependent recombination generates exclusively double exchanges at both sides of the marker. (2) Marker-dependent recombination is highly sensitive to DNA base sequence at the site of the marker and to that at the corresponding site on the chromosome of the other parent. (3) Within certain limits, the contribution of the marker-dependent mechanism to the total recombination frequency is distance-independent and thus constitutes a constant component. YBRID DNA molecules with complementary strand sections derived from H different parents are generally believed to arise in genetic recombination. A genetic marker falling within such a hybrid region generates a mismatch that might be repaired. The most convincing support for the notion of mismatch repair comes from studies on co-conversion in the fungi Ascobolus immersus (LEBLON and ROSSIGNOL 1973) and Schizosaccharomyces pombe (GUTZ 1971), and from experiments on transfection by DNA heteroduplexes with mismatches at four sites in phage h (WAGNER and MESELSON 1976). In T4 phage, some genetic phenomena such as high negative interference (CHASE and DOERMANN 1958) and ultrafine-structure map expansion (TESSMAN 1965), as well as the loss of heteroduplex heterozygotes after prolonged incubation of phage-infected Escherichia coli cells under nonreplicating conditions (BERGER and PARDOLL 1976), can be interpreted as originating from mismatch repair. However, evidence for the involvement of mismatch repair in T4 general recombination remains scanty. The length of a single-strand DNA segment excised upon the repair of Genetics 102: 615-625 December, 1982 616 V. P. SHCHERBAKOV ET AL. mismatched rrr mutations in normal T4 phage crosses seems not to exceed about 10' bases, whereas the maximum frequency of recombination attributable to mismatch repair is on the order of IOp3 (TOOMPUU and SHCHERBAKOV 1980). This permits us to focus on recombination frequencies in the range of to lop3 when specific manifestations of mismatch repair are sought in formal recombination analysis. Proceeding from this consideration, we carried out a large series of twoand three-factor crosses between closely linked T4 rrr mutants. Six of the 20 markers studied were found to be susceptible to an extra recombination mechanism to which the other markers were much less susceptible. Below is a summary of several experimental approaches resulting in evidence for mismatch repair in marker-dependent recombination, whereas the companion paper ( SHCHERBAKOV et al. 1982) deals specifically with quantitative implications of the phenomenon. MATERIALS AND METHODS Bacteriophages: The d l mutants of T4 phage used in this study are shown in Figure 1. The wildtype T4D strain was kindly supplied by K. EBISUZAKI; the mutants HE122, FCI, UV375, 360, X511, FC9, FCO, UV357, FC21, FC40, and UV200 were obtained as a generous gift from S. P. CHAMPE; the mutant H72 was a contribution of Yu. P. VINETSKI. The remaining mutants were isolated by us. The opal mutants op360 and opUV357 were derived from the ocher mutants 360 and UV357, respectively, as a result of spontaneous transitions (ocher 4 opal) selected by plating on an opal-suppressing E. coli strain. Similarly, the amber mutants amUV200, omUV375, am360 and amUV357 were derived from the appropriate ocher mutants as a result of spontaneous transitions (ocher -+ amber) selected by plating on an amber-suppressing E. coli strain. The plus frameshift mutations 81 and p8 and the minus frameshift mutations /39 and P I 0 were selected as intragenic suppressors of FC1 and P8, respectively, whereas the plus mutation fill was selected as an intragenic suppressor of P9. In all cases in which the mutations were isolated in a T4B background, they were made isogenic by incorporating them into a T4D background by six backcrosses against T4Dr+ at multiplicities of mutant and wild-type phages equal to one and nine, respectively. The mutations were aligned on the basis of twoand three-factor cross data. The sequence of the mutants HE122, FC1, UV375,360, X511, FC9, FCO, UV357, FC21, and FC40, as well as of the barriers (terminating codons located out of phase) bl (ocher), bL (opal), and b3 (ocher), agrees with that in the maps of BARNETT et al. (1967) and KATZ and BRENNER (1975). Bacteria: E. coli strains BB, 594(X), K223(h), CA167(X) (received from the Institute of Genetics of Microorganisms, Moscow), CA244(h), CA180(X). and CA265(X) (a generous gift of F. W. STAHL) were used. Strain BB was used as a host in all phage crosses, in preparing phage stocks, in phage titration, and in measuring the total phage yield in the crosses. Strains 594(h)suand CA244(X)suare nonpermissive for all rII mutants. Strain K223(X)sutis permissive for rrr mutants of the opal type and nonpermissive for other rfI mutants. Strain CA167(h)su$ is permissive for ocher and amber mutants and nonpermissive for other rII mutants. All the strains used are permissive for T4r+. Media: L broth: 1% Difco tryptone, 0.5 % Difco yeast extract, 0.1% glucose, and 1% sodium chloride, pH 7.0; used for growing liquid E. coli cultures and for standard crosses. Beef-peptone broth: 5% Hottinger broth, 0.5% sodium chloride, and 1% bactopeptone (Spofa), pH 7.0; used for weekly E. coli cultures. Peptone broth: 1% bactopeptone (Spofa), 0.8% sodium chloride, 0.2% sodium citrate, and 0.1% dextrose, pH 7.0; used for phage dilution. Beef extract agar: 5% Hottinger broth, 0.5% sodium chloride, 1% bactopeptone (Spofa), and 2% Difco agar; used for E. coli slant cultures. Peptone agar: 1% bactopeptone (Spofa), 0.8% sodium chloride, and 2% agar (U.S.S.R.), pH 7.0; used for phage plating (bottom agar). Soft agar D: 1% Difco tryptone, 0.23% Difco yeast extract, 0.47% glucose, 0.77% sodium chloride, and 0.7% Difco agar, pH 7.0; used for phage plating (top agar). Management of bacterial cultures: A colony of the bacterial strain was transferred to a beef extract agar slant and grown for 24 hr at 33'. Bacteria from one slant were suspended in 10% sucrose RECOMBINATION IN T4 PHAGE 617