ABSTRACT
In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism - RecA-mediated excision repair - in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.
Subject(s)
DNA Repair , DNA, Bacterial/biosynthesis , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Rec A Recombinases , Culture Media , DNA Damage , DNA, Bacterial/genetics , Endodeoxyribonucleases/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Plasmids/genetics , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Transformation, BacterialABSTRACT
Lesions that transiently block DNA synthesis generate replication intermediates with recombinogenic potential. In order to investigate the mechanisms involved in lesion-induced recombination, we developed an homologous recombination assay involving the transfer of genetic information from a plasmid donor molecule to the Escherichia coli chromosome. The replication blocking lesion used in the present assay is formed by covalent binding of the carcinogen N-2-acetylaminofluorene to the C8 position of guanine residues (G-AAF adducts). The frequency of recombination events was monitored as a function of the number of lesions present on the donor plasmid. These DNA adducts are found to trigger high levels of homologous recombination events in a dose-dependent manner. Formation of recombinants is entirely RecA-dependent, the RecF and RecBCD sub-pathways accounting for about 2/3 and 1/3, respectively. Inactivation of recG stimulates recombinant formation about five-fold. In a recG background, the RecF pathway is stimulated about four-fold, while the contribution of the RecBCD pathway remains constant. In addition, in the recG strain, a recombination pathway that accounts for about 30% of the recombinants and requires genes that belong to both RecF and RecBCD pathways is revealed.
