In vitro inhibition of RecA-mediated homologous pairing by UmuD'C proteins.
The process of SOS mutagenesis in Escherichia coli requires: i) the replisome enzymes; ii) RecA protein; and iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. It has recently been shown that the UmuD'C complex, if overproduced, inhibits recombinational repair of a UV-damaged plasmid DNA as well as homologous recombination in an Hfr x F- cross. Since UmuD'C proteins might inhibit an early recombination step by interacting with a RecA nucleo-protein filament, we checked whether UmuD'C proteins will inhibit RecA promoted homologous pairing in vitro. We tested the inhibitory action of UmuD'C proteins in a crude bacterial extract containing possible cofactors such as chaperone proteins that ensure the proper folding of UmuC and the assembly of the UmuD'C complex in vivo. We used a novel recombination assay in which RecA protein promotes the formation of a stable plectonemic joint between a circular single-stranded DNA immobilized onto a membrane and an incoming homologous linear duplex DNA. Under these conditions we show that UmuD'C proteins inhibit the formation of joint molecules.
Szpilewska
H
H
Groupe d'Etude Mutagénèse et Cancérogénèse, Institut Curie, Paris, France.
Bertrand
P
P
Bailone
A
A
Dutreix
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D009876:Operon; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D001426:Bacterial Proteins; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli