Biochemical basis for the essential genetic requirements of RecA and the β-clamp in Pol V activation

Abstract

In Escherichia coli, it is genetically well established that the ␤-clamp and RecA are essential cofactors that endow DNA polymerase (Pol) V with lesion bypass activity. However, the biochemical basis for these requirements is still largely unknown. Because the process of translesion synthesis (TLS) requires that the specialized DNA polymerase synthesize in a single binding event a TLS patch that is long enough to resist external proofreading, it is critical to monitor Pol V burst synthesis. Here, we dissect the distinct roles that RecA and the ␤-clamp perform during the Pol V activation process using physiologically relevant long single-stranded template DNA, similar to those used in genetic assays. Our data show that the ␤-clamp endows the complex between Pol V and the template DNA with increased stability. Also, the RecA filament formed in cis on the single-stranded DNA produced downstream from the lesion stretches the template DNA to allow smooth elongation of the nascent strand by Pol V. The concurrent action of both cofactors is required for achieving productive TLS events. The present article presents an integrated view of TLS under physiologically relevant conditions in E. coli that may represent a paradigm for lesion bypass in other organisms. When a replication apparatus encounters a replicationblocking lesion in one of the template strands, the replicative helicase keeps opening the parental duplex, replication continues on the undamaged strand, and a gap is formed in the damaged chromatid opposite the lesion (1, 2). Functional uncoupling of the leading and lagging strand DNA polymerases has been observed both in vivo (3) and in vitro (4, 5). The singlestranded gaps formed opposite the lesions are converted into the molecular signal for the SOS response on formation of a single-stranded ssDNA-RecA filament. Translesion DNA synthesis (TLS) that is largely mediated by DNA polymerase (Pol) V (encoded by the umuDC operon) requires RecA (6-10) and the ␤-clamp, the replication processivity factor (11). In vivo, RecA filament formation is a complex reaction that involves the recombination mediator proteins RecFOR to counteract the high propensity of ssDNA-binding (SSB) protein to bind ssDNA (12). Indeed, RecFOR proteins are essential for efficient Pol V-mediated TLS and mutagenesis both in vitro and in vivo (13). Naked regions of ssDNA are unlikely to persist in vivo even for short time periods as shown during lagging-strand replication (14). Because Pol V-mediated TLS is induced late during the SOS response, the ssDNA gaps generated downstream the replication-blocking lesions are initially covered by SSB and gradually replaced by RecA in a RecFOR mediated reaction path. It is, thus, sensible to include these proteins in Pol V-mediated TLS reconstitution experiments as we showed previously (13). The mechanism by which RecA activates Pol V has been a matter of debate over the years (15), and we will briefly discuss it in the light of a recently published article (16).TLS entails at least two DNA polymerase swi...

Translesion DNA Synthesis and Mutagenesis in Prokaryotes

“…Interestingly in 2012, the same group published a paper showing that Pol V lesion bypass can be mediated by either cis-or transRecA filaments (Karata et al 2012). Using a single-stranded circular substrate, Pol V was shown to bypass a single TT-CPD lesion in the presence of both the b-clamp and a cisRecA filament in good agreement with previously published data Fujii and Fuchs 2009). On the other hand, efficient lesion bypass was also possible when Pol V was activated with trans-RecA provided ATPgS was added to stabilize the trans-RecA filament.…”

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

“…Our observations cannot rule out alternative models, however, including a scenario in which UmuDЈ 2 mediates interactions with UmuC and RecA or in which competition for DNA substrates forms the basis of Pol V-induced inhibition of homologous recombination. Disruption of the interaction of Pol V with RecA could lead to the UV sensitivity that we observed, as that interaction is critical for TLS (23,31,58,60,61). In sum, we show here that the N terminus of loop 1 plays an essential role in cellular resistance to DNA-damaging agents and facilitates possible interactions with RecA that could be critical for Pol V-mediated UV mutagenesis.…”

“…In addition to the roles of RecA in the induction of the SOS response and the cleavage of UmuD, RecA also plays a role in the activation of Pol V for TLS (23,31,58,60,61). While the exact mechanism of Pol V stimulation by RecA remains to be determined, it is understood that RecA is required for Pol V mutagenesis.…”

“…The ␤ clamp substantially increases processivity in both Pol III and Pol IV in E. coli but increases the processivity of Pol V only 3-to 5-fold (25,35,42,80). The ␤ clamp interacts directly with UmuC utilizing a canonical (4,6,20,41,73) ␤-binding motif, an interaction that is critical for UmuC to participate in TLS (4,6,73).In addition to the roles of RecA in the induction of the SOS response and the cleavage of UmuD, RecA also plays a role in the activation of Pol V for TLS (23,31,58,60,61). While the exact mechanism of Pol V stimulation by RecA remains to be determined, it is understood that RecA is required for Pol V mutagenesis.…”

“…Opposite C 8 -dG-acetylaminofluorine, Pol V inserts dA (3,23,24); Pol V bypasses N 2 -benzo[a]pyrene-dG with relative inaccuracy, while it bypasses N 6 -benzo[a]pyrene-dA fairly accurately (37,63,83).…”