Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase

Abstract

All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the ␤-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered.DNA repair ͉ DNA replication ͉ replication fork ͉ replisome sliding clamp C hromosomes are duplicated by replisome machines containing helicase, primase, and DNA polymerase activites (1). Replicative DNA polymerases are tethered to DNA by ringshaped sliding clamp proteins. The Escherichia coli polymerase III (Pol III) holoenzyme replicase contains 10 different subunits consisting of a clamp loader that binds 2 molecules of Pol III, each tethered to DNA by the ␤-sliding clamp for processive leading and lagging strand synthesis (2). Pol III also binds tightly to the hexameric DnaB helicase that encircles the lagging strand template. The Pol III-to-DnaB interaction is mediated by the -subunit within Pol III (3). This tight connection couples Pol III motion to DnaB unwinding and increases the rate of unwinding by DnaB helicase from a basal level of 35 bp/s to a rate in excess of 500 bp/s (3). These tight connections of Pol III to DnaB and the ␤-clamp provide the replisome with sufficient stability, in principle, to replicate the entire genome. In practice, however, replication of long chromosomal DNA is an uneven path with a variety of obstacles along the way, including protein blocks and damaged templates.In the presence of high levels of DNA damage, Ͼ40 gene products are induced by the SOS response that act to restore genomic integrity and assure cell survival (4). Among the SOS-induced gene products are 3 translesion DNA polymerases, Pols II, IV, and V (encoded by polB, dinB, and umuCD, respectively), that perform potentially mutagenic DNA synthesis across template lesions (5). Pols IV and V are members of the Y family of error-prone DNA polymerases; they lack a proofreading 3Ј-5Ј exonuclease (6, 7). Pol II is a B-family polymerase; it has high fidelity and contains proofreading activity (8). Pols II and IV are present during normal cell growth and may be involved in repairing low levels of DNA damage that occur routinely in the cell (9-11). Pol V, however, is closely associated with mutagenesis and is not detectable in cells under normal conditions (7,12,13).Pol II and Pol IV are among the first gen...

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

“…We previously showed that TLS Pols II and IV exchange with Pol III* during replisome progression, although RecA was not present in those studies (2). According to our recent findings, one may presume that, once RecA has been induced, Pol III* is no longer capable of function.…”

“…Both in vivo and in vitro studies indicate that TLS Pols can switch with the Pol III replicase during replication and that the switching process mainly occurs by simple mass action (2,15,17). Although mass action may be an important determinant, most likely the process is more complex (14,38).…”

“…In Escherichia coli, TLS Pol II and TLS Pol IV are present in normal growing cells along with the high-fidelity chromosomal replicase, Pol III. Previous studies have shown that all three Pols function at replication forks with DnaB helicase, primase, the β sliding clamp, and the clamp loader (2). Bulk chromosome replication is performed by Pol III, and the mechanism that regulates the access of TLS Pols to the replisome is largely unexplored.…”

“…Upon DNA damage, the induced levels of TLS Pols take over the cellular replicase (2,14,15). Our previous studies have shown that Pols II and IV replace Pol III within the replisome without causing fork collapse, as they preserve the DNAbound helicase and sliding clamp to form TLS Pol replisomes (2).…”

“…During normal chromosome duplication, the intracellular levels of Pols II and IV are not sufficient to take over Pol III-based replisomes (2). Upon DNA damage, the induced levels of TLS Pols take over the cellular replicase (2,14,15).…”