Lesion Bypass by the Escherichia coli DNA Polymerase V Requires Assembly of a RecA Nucleoprotein Filament

Abstract

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOSregulated proteins UmuD and RecA and the singlestrand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATP␥S, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.Genomic DNA is afflicted by numerous lesions that might interfere with its propagation and with gene expression (1). Most of these lesions, which are usually base modifications, are repaired by cellular DNA repair mechanisms (1). When the replication fork encounters a blocking DNA lesion that has escaped repair, replication stops forming a ssDNA 1 region in DNA (2). In Escherichia coli at least two mechanisms, which are regulated by the SOS response (3), act to repair the gap by converting the ssDNA region into a dsDNA region without actually removing the damaged nucleotide. Recombinational repair patches the gap with a complementary DNA segment from the fully replicated sister chromatid (4, 5), whereas translesion replication fills in the gap by DNA synthesis. This pathway, also termed lesion bypass or error-prone repair, is mutagenic, because DNA lesions often cause misincorporation by DNA polymerases, leading to the formation of mutations (2, 6).The in vitro reconstitution of SOS translesion replication with purified components (7-9) established that SOS-targeted mutagenesis occurs by replication through DNA lesions by DNA polymerase V (UmuC) 2 in the presence of UmuDЈ, RecA, and SSB (10, 11). Pol V effectively bypasses a synthetic abasic site (10, 11), a cyclobutyl TT dimer and a 6-4 TT adduct (12), leading to targeted mutations. When replicating an undamaged DNA template pol V is highly mutagenic and forms preferentially purine-purine and pyrimidine-pyrimidine mismatches, resulting in transversion mutations (13). These activities of pol V are responsible for SOS mutagenesis targeted to DNA lesions and for untargeted mutagenesis, which occurs in undamaged DNA regions.Proteins similar to UmuC are widespread from E. coli...

DNA Damage Control by Novel DNA Polymerases: Translesion Replication and Mutagenesis

“…In a TLR system based on a gapped plasmid, lesion bypass was found to be inhibited by ATP␥S, consistent with the notion that progression of pol V causes disassembly of the RecA filament. The inhibitory effect of ATP␥S on bypass by pol V was milder than the effect on initiation (66). These results suggest that once loaded on the primer-template at the initiation stage, the binding of pol V to DNA is strongly stabilized such that it can displace RecA from DNA, aided by SSB, even in the presence of ATP␥S.…”

“…1, step 2) (66). This filament, which assembles in the 5Ј33Ј direction, covers the ssDNA region and continues to the dsDNA region adjacent to the ssDNA region, including the primer terminus near the lesion (66).…”

“…In the subsequent step of initiation (Fig. 1, step 3), pol V binds to the primer-template, guided by the RecA filament, which serves as a targeting platform (66,67). Loading of pol V onto the primertemplate is stabilized by at least three known protein-protein and DNA-protein interactions.…”

“…1, step 3). This suggestion is based on the strong inhibition of initiation of TLR caused by ATP␥S, a poorly hydrolyzable ATP analog that strongly stabilizes RecA binding to DNA (66). This strong binding presumably inhibits displacement of RecA from the primer-template region by pol V. SSB is likely to help pol V in displacing RecA from DNA (70).…”

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

“…This ''minimal'' p͞t DNA is nevertheless long enough to allow formation of a RecA filament (26). The presence of a RecA filament proximal to a template damage site appears to be a requirement for pol V-catalyzed TLS lesion (12,26,35).…”