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. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.
Institute of Problems of Chemical Physics RAS, Chernogolovka, Moscow Region, Russia. svp@icp.ac.ru
Victor P
VP
Shcherbakov
Lidiya
L
Plugina
Tamara
T
Shcherbakova
Svetlana
S
Sizova
Elena
E
Kudryashova
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004260:DNA Repair; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D004706:Endodeoxyribonucleases