Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations.

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.

[Gene rIIB from bacteriophage T4 in genetic recombination]. / Gen rIIB bakteriofaga T4 v geneticheskoi rekombinatsii.

The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied. Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants. In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation. In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene. The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically. In the third series, all crosses were intragenic (rIIb x rIIb). In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background. The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene. The possible role of the rIIB protein in genetic recombination is discussed.

Marker-dependent recombination in T4 bacteriophage. IV. Recombinational effects of antimutator T4 DNA polymerase.

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3'-->5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49)-->3'-->5' exonuclease (gp43)-->DNA polymerase (gp43)-->DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.

Genetic recombination in bacteriophage T4: single-burst analysis of cosegregants and evidence in favor of a splice/patch coupling model.

To reveal the structure of penultimate DNA intermediates in T4 bacteriophage recombination, resolution of which produces free recombinant molecules, a single-burst analysis of the recombinant progeny was made in multifactor crosses, enabling one to determine quantitatively the different recombinants generated by one or two exchanges within the same chromosome segment. It was found that double and single exchanges are highly correlated in T4 recombination. These results were interpreted as evidence for simultaneous formation of a splice/patch pair as the primary recombination products. A recombination model called here the "splice/patch coupling model" is presented according to which resolution of a single DNA intermediate results in two linear heterozygous molecules containing a patch and a splice, respectively, in homologous positions.

Marker-dependent recombination in T4 bacteriophage. III. Structural prerequisites for marker discrimination.

Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.

Marker-dependent recombination in T4 bacteriophage. I. Outline of the phenomenon and 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.

Marker-dependent recombination in T4 bacteriophage. II. The evaluation of mismatch repairabilities in crosses within indicator distances.

The contribution of mismatch repair to genetic recombination in T4 phage has been evaluated by three independent approaches: (1) testing for non-additivity of recombinant frequencies; (2) measurements of double exchange frequencies in three-factor crosses: (3) comparisons of recombination abilities of mutations occupying the same site. Quantitative agreement among the results of these approaches suggests that within distances much less than the mean length of hybrid regions, mismatch repair accounts perfectly for high negative interference as measured in three-factor crosses and as manifested by non-additivity in two-factor crosses. The mismatch repair mechanism readily recognizes only particular mismatches, the repair frequency being dependent on the base sequence in both strands of the mismatched region.

[Recombination in bacteriophage T4: the nature of the disturbances introduced into genetic recombination by a region of non-homology]. / Rekombinatsiia u bakteriofaga T4: o prirode vozmushchenii, vnosimykh v geneticheskuiu rekombinatsiiu oblast'iu negomologii.

Effects of a non-homology region associated with a deletion marker on recombination were studied in two-factor crosses between rII mutants of T4 phage. The basic experimental approach consisted of the comparison of a deletion with the point mutation located at one of the borders of the segment deleted in crosses against the same set of the test markers. The results obtained are in good accordance with the predictions of the model of elastic interaction of migrating cross connection with the nonhomology region. This accordance argues strongly in favour of the hybrid region propagation via branch migration in T4 recombination intermediates.

["Silent" marker effect in T4-phage recombination]. / Effekt "molchashchego" markera v rekombinatsii u faga T4.

Effects of the silent sites of heterozygosity on genetic recombination have been studied in crosses between the markers localized in the proximal segment of rIIB gene of T4 phage. It was found that silent markers could inhibit as well as stimulate genetic recombination. For the expression of these marker effects, the distance between the silent marker and those for which recombination was being monitored, appeared to be critical. There were no marker interference in the crosses in which an influencing marker was separated from recombining ones by a distance exceeding the length of DNA segment involved in single mismatch-repair event. The most prominent marker effects were observed when an influencing marker and recombining one were linked closely enough to form a mutual mismatched region. From the results obtained it is evident that the target which is recognized by mismatch-repair mechanism is formed from DNA strands originating from both parents, and both strands of the heteroduplex are essential for the recognition.

[Contribution of correction to genetic recombination in T4-phage measured in 3-factor crosses over indicator distances]. / Vklad korrektsii v geneticheskuiu rekombinatsiiu u faga T4, izmeriaemyi v trekhfaktornykh skreshchivaniiakh na indikatornykh rasstoianiiakh.

Marker-dependence of recombination in T4 phage was studied in crosses over indicator distance between the markers localized in the short proximal segment of rIIB gene. In three-factor crosses a-b+c- x a+b-c+, the frequencies of recombinants arising as a result of correction of the central marker b to the wild type allele (chi b leads to +) have been determined. The measured values chi b leads to + were found to be in good agreement with the analogous values determined in two-factor crosses. This agreement suggests that in T4 crosses over indicator distances only coupled double exchanges (conversion-like events) are marker-specific and all single exchanges are marker-independent.

[Formal genetic study of the enzymology of T4 phage recombination. I. Effect of the antimutational T4 DNA-polymerase on noncorrection-type recombination]. / Formal'no-geneticheskoe izuchenie énzimologii rekombinatsii u faga T4. Soobshchenie I. Vliianie antimutatornoi DNK-polimeraxy T4 na rekombinatsiiu nekorrektsionnogo tipa.

Effect of temperature sensitive antimutator L42 allele of gene 43 of T4 phage on the parameters of non-correction-type mechanism of genetic recombination have been studied under permissive conditions. By comparison of a point mutant with a deletion one, the critical frequency R (xi) corresponds to recombination between two markers separated by a distance equal to mean genetic length of the hybrid region was measured to be 4.15-10(-2). This value exceeds the frequency R (xi) determined with the parallel data in normal (43+) crosses by a factor of 1.6. The length of the hybrid region was shown not to change in the presence of L42 allele. The frequency of formation of the hybrid regions with recombinant flanks and of those with parental flanks increases in the presence of L42 by a factor of 1.3 and 1.8 respectively.

[Simple method of determining the R(xi) parameter of a half-chromatid chiasma]. / Prostoi sposob opredeleniia parametra R(xi) polukhromatidnoi khiazmy.

Comparison of a point mutant of T4 phage with a deletion one in two-factor crosses against other point mutants demonstrated that at equal distances the deletion marker gave constantly lower frequencies of wild-type recombinants than the point marker did. The pattern of the distance dependence of this difference in the frequencies of recombinants agrees well with the hypothesis that mismatched regions formed by deletion markers undergo repair to the mutant allele with a probability close to unity. The equations have been obtained, connecting the frequency of the wild-type recombinants in a cross between two point mutants with the analogous frequency in a cross between a deletion marker and a point one. Analysis of the experimental data by means of these equations enabled the critical frequency R (xi) (recombination frequency between two point mutants separated by a distance equal to the mean length of hybrid region) to be measured in two independent ways.

[Morphologico-genetic study of the enzymology of T4 phage recombination. II. Effect of antimutational T4 DNA-polymerase on correction-type recombination]. / Formal'no-geneticheskoe izuchenie énzimologii rekombinatsii u faga T4. Soobshchenie II. Vliianie antimutatornoi DNK-polimerazy T4 na rekombinatsiiu korrektsionnogo tipa.

Effect of temperature antimutator L42 allele of gene 43 of T4 phage on the parameters of correction-type mechanism of genetic recombination have been studied under permissive conditions. The effect of L42 was found to be marker- as well as distance-dependent, and the pattern of these dependences shows that in the presence of antitumor DNA polymerase the distribution of the length of the repair region becomes more sharp and the mean length of the repair region becomes shorter. From this, it is concluded that T4 DNA polymerase is directly involved in the repair of mismatched region in recombinational intermediates, excising non-matched DNA strand and then filling the gap arisen.

[Asymmetry in the frequencies of reciprocal recombinants in crosses of T4 phage rIIB mutants]. / Asimmetriia chastot retsiproknykh rekombinantov v skreshchivaniiakh mutantov rIIB faga T4.

The frequencies of reciprocal recombinants in crosses between rIIB mutants of T4 phage were shown to differ from each other. In terms of the correction model, this asymmetry of genetic recombination was used to measure the comparative correctability of the mismatched regions to the wild type and to the mutant alleles. The data obtained are in quantitative agreement with the analogous values for the same mismatched regions determined by comparison of the markers located at the same site. This strongly suggests that the asymmetry of genetic recombination in T4 reflects the corresponding difference in rates of correction of the mismatched regions in heteroduplexes in opposite directions.

[Allele specificity of genetic recombination in T4 phage using the indicator crossing study method]. / Issledovanie allel'-spetsifichnosti genticheskoi rekombinatsii u faga T4 metodom indikatornykh skreshchivanii.

The diagrams of relative correction ability of eighteen rII mutants of T4 phage were constructed on the basis of two-factor crosses, which were grouped into indicator series. In each series a pair of closely linked compared markers was crossed against indicator ones, the latter being distant enough so as to avoid simultaneous correction with the compared marker. The differences between the frequencies of wild type recombinants in crosses of two compared markers with indicator ones remained constant within the series and can be used as a measure of the differences between the compared markers in their correction ability. Mutants of base substitution type have small but statistically significant differences in correction ability. Simultaneous substitution of two bases in one codon yields a mutant which shows higher correction ability when compared to the mutant obtained as a result of substitution of only one base in the same codon. Frame shift mutants show much wider range of correctibility: some of them are corrected more rarely and others more frequently than base substitution mutants are.

[Contritution of correction to genetic recombination in T4 phage measured by the effect of map contraction]. / Vklad korrektsii v geneticheskuiu rekombinatsiiu u faga T4, izmeriaemyi po éffektu suzheniia karty.

Marker-dependence of the fine structure map contraction in T4 phage is studied in two-factor crosses between rIIB mutants separated by indicator distances. The genetic intervals, which were short as compared with mean length of the heteroduplex region in hybrid DNA molecules but which exceeded the length of the DNA strand involved in a single correction event, were selected as indicator ones. On the basis of a deviation of measured frequencies from additivity (map contraction) the marker-specific frequencies of wild type recombinants arising as a result of correction to the wild type (kappa (- leads to +)) were calculated. For the most of the marker studied both of the base substitution and frame shift type the frequencies kappa (- leads to +) have the values below 2.10(-4). In the case of three most highly corrected frame shift markers with kappa (- leads to +) being 14.10(-4)--17.10(-4), about ten percent of all mismatched regions are corrected to the wild type.