Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coil by the SOS-induc ible DNA polymerase V (UmuC), an error-prone polymerase, which is specializ ed for replicating through lesions in DNA, leading to the formation of muta tions. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro as say system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA n ucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for si ngle-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate t he assembly of the RecA nucleoprotein filament; however, it has at least on e additional role in lesion bypass. ATP gammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activ ity. Lesion bypass does not require stoichiometric binding of UmuD' along R ecA filaments. In summary, the RecA nucleoprotein filament, previously know n to be required for SOS induction and ho mologous recombination, is also a critical intermediate in translesion replication.