Double-strand break repair and recombination-dependent replication of DNA in bacteriophage T4 in the absence of UvsX recombinase: replicative resolution pathway.

The effects of mutations in bacteriophage T4 genes uvsX and 49 on the double-strand break (DSB)-promoted recombination were studied in crosses, 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×ets1 and in three-factor crosses of the type i×ets1 a6, where ets1 is an insertion in the rIIB gene carrying the cleavage site for SegC; i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site, and a6 is rIIA point mutation located at 2040 bp from ets1. The frequency/distance relationships were obtained in crosses of the wild-type phage and of the amber mutant S17 (gene uvsX) and the double mutant S17 E727 (genes uvsX and 49).

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4.

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49.

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants.

Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing.

Double-strand break repair in bacteriophage T4: coordination of DNA ends and effects of mutations in recombinational genes.

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair.

Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks.

A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC(+) conditions, the ets1 behaves as a recombination hotspot.