Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Abstract

npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer. DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…These bulky lesions distort the double helix and block the progress of DNA replication polymerases. The prolonged stalling of replication forks can result in further damage from collapsed replication forks and incomplete replication, ultimately causing cell death [Saleh-Gohari et al, 2005;Andersen et al, 2008]. To avoid this, DDT mechanisms are activated, whereby translesion polymerases allow bypass synthesis across bulky lesions Zhang et al, 2011;Sale et al, 2012].…”

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…PCNA can stimulate DNA synthesis by pol ζ [22]. There is evidence that a Rev3-Rev7-Rev1 complex associates with monoubiquitinated-PCNA through ubiquitin-binding motifs in REV1, which helps to enable pol ζ or other REV1-interacting bypass polymerases to insert a base opposite damage and then extend from the resulting non-standard primer-template [22,24] (see the review by Andersen et al in this issue [25]). One current model for how yeast pol ζ and Rev1 may function at a stalled replication fork is shown in Figure 1.…”

The DNA Polymerase Activity ofSaccharomyces cerevisiaeRev1 is Biologically Significant

“…The mechanism of action and identities of the participating proteins are not fully understood, but a particular modification on PCNA is instrumental in the process. Whereas TLS is associated with the monoubiquitination of PCNA by Rad6/Rad18, the error-free tolerance branch relies on the Lys-63 linked polyubiquitination of PCNA (Andersen et al 2008). This polyubiquitination of PCNA is completed through the action of Rad5 and the Mms2/Ubc13 heterodimer.…”