Abstract
A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3' → 5' proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its "little finger" domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions.
Highlights
Translesion synthesis DNA polymerases insert nucleotides opposite bulky template lesions not tolerated by replicative DNA polymerases
Bypass of DNA template damage by translesion synthesis is an essential survival mechanism for all organisms, allowing DNA replication to proceed past the point of damage so that the cell cycle can be completed and cell division accomplished, even at the expense of the misincorporated nucleotides that can be inserted opposite the damage by the generally error-prone TLS DNA polymerases
Some DNA lesions are corrected by either the base excision or nucleotide excision repair pathways either pre- or postreplicatively; it seems likely that occasions arise with some frequency that require lesion bypass coincident with DNA replication
Summary
Translesion synthesis DNA polymerases insert nucleotides opposite bulky template lesions not tolerated by replicative DNA polymerases. Lesions encountered on the leading strand template cause replisome stalling and lesion bypass may occur via different mechanisms: (a) dissociation (or perhaps enzymatic removal) of the replication machinery at the stalled fork followed by modulation of the fork structure, repair of the lesion, and subsequent replication restart via origin-independent reloading of the replisome [3, 4]; (b) lesion skipping, where the replisome only stalls transiently at the lesion and via a primasecatalyzed leading strand priming event downstream, resumes coupled leading and lagging strand synthesis, jumping over the lesion [5], leaving both the lesion and a gap in the nascent leading strand behind as substrates for daughter strand gap repair [6]; and (c) translesion synthesis (TLS) DNA polymerases could exchange with the blocked replicative polymerase to bypass the lesion, yielding fully replicated DNA that still contains the template damage Nonmutagenic repair pathways, such as nucleotide excision repair, could excise and repair the lesion postreplicatively. Pol IVcatalyzed TLS came at the expense of leading strand lesion skipping, suggesting that it may be the preferred reaction at stalled replication forks
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