[In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4].

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC+ x i ets 1 segCDelta crosses, where ets 1 is an insertion in the initial part of the rII gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets 1, and segCDelta is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets 1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets 1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i x i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets 1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.

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.

[The structural characteristics of the polyribosomes in the rat liver studied by means of a tritium label]. / Osobennosti struktury poliribosom pecheni krys, izuchennye metodom tritievoi metki.

Polyribosomes isolated from the rat liver in a medium with low ionic strength were irradiated by "hot" tritium atoms under conditions providing for the replacement of the hydrogen atoms located at the surface of polyribosomes by tritium. After fractionation of such polyribosomes, the radioactivity of the obtained fractions was measured and their proportions were calculated for the total surface accessible for the tritium atoms (in %), as well as their specific radioactivity. The material loosely associated with the polyribosomes and containing amino acyl-tRNA-synthetases is more radioactive than rRNA and r-proteins, especially concerning their specific radioactivity. This suggests that the material is organized as individual molecules located on the surface of ribosomes. The specific radioactivity of the RNA-component of this material (tRNA) is twice that of proteins, thus suggesting its surface localization in the composition of loosely associated material. Based on the pattern of labeling of rRNA and r-proteins of the native and preliminarily dissociated polyribosomes, we propose that the material, loosely associated with the polyribosomes, has affinity to both rRNA and r-proteins.

[Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid]. / Poliribosomy, izolirovannykh iz pecheni krys v srede s nizkoi ionnoi siloi, sposobny k avtonomnoi transliatsii v beskletochnoi sisteme bez dobavleniia kletochnogo soka.

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.

[Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide]. / Dinamika biosinteza komponentov beloksinteziruiushchego apparata pecheni krys na étape vosstanovleniia transliatsii, ingibirovannoi tsiklogeksimidom.

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are 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.

[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.

[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.

[Suppression of DNA-ligase deficiency and selective breeding of mutants and recombinants of T4 phage with phenotype rII]. / Supressiia nedostatochnosti po DNK-ligase i selektivnyi otbor mutantov i rekombinantov s fenotipom rII u faga T4

A number of methods which greatly simplify the introduction of frame shift mutations in the limited segment of the phage T4 rII genes were developed for studying genetical recombination between closely linked markers. These methods enable to construct multiple rII mutants with relatively small expenditure of labour. Suppression of gene 30 deficiency by rII mutations has been studied for a variety of conditions. The results obtained indicate that rIIB polypeptide is involved in two different activities: rIIB region which is dispensable for growth on lambda-lysogenic Escherichia coli strains behaves as indispensable in phenomenon of suppression of ligase deficiency.