Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition.
Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.
Sommer
Suzanne
S
Institut de Génétique et Microbiologie, Bât. 409, Université Paris-Sud, F-91405, Orsay, France. suzanne.sommer@igmors.u-psud.fr
Becherel
Olivier J
OJ
Coste
Geneviève
G
Bailone
Adriana
A
Fuchs
Robert P P
RP
D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007266:Inhibition, Psychological; D009154:Mutation; D010957:Plasmids; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic; D016944:Sulfolobus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D004247:DNA; D011938:Rec A Recombinases; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase