DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Abstract

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase , the POLH gene product. A deficiency in DNA polymerase due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase cooperates with DNA polymerases and to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases and , but not , protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.carcinogenesis ͉ DNA repair ͉ lesion bypass ͉ replication ͉ ultraviolet T LS is a fundamental mechanism for tolerating DNA damage that has escaped repair, carried out by specialized lowfidelity DNA polymerases, which synthesize across a wide variety of DNA lesions (1). At least 5 TLS DNA polymerases are present in mammals, four of which, DNA polymerases , , , and REV1, belong to the Y superfamily. The fifth TLS polymerase is pol, which belongs to the B family, and is the only TLS polymerase known to be essential in mammals (2-5). TLS polymerases exhibit a certain degree of specificity for their substrate DNA lesions, and their activity is tightly regulated (6-9). The most well characterized TLS polymerase is pol, which is specialized to bypass cyclobutane pyrimidine dimers (CPD) in a relatively error-free manner. The biological significance of pol is illustrated by the hereditary disease xeroderma pigmentosum variant (XPV), in which germ-line mutations in the POLH gene, encoding pol, cause an extreme 1000-fold increased predisposition to sunlight-induced skin cancer (10, 11). Cells from XPV patients exhibit a slightly increased UV sensitivity, and a dramatic UV hypermutability (12), which is responsible for their extreme cancer predisposition. The UV hypermutability is explained by the activity of a back-up DNA polymerase that performs TLS across CPD with lower efficiency and higher error-frequency. Although there is evidence that pol is involved in TLS across CPD in XPV cells (13-15), additional polymerases may be involved, and the picture is far from being complete. This is an important issue because these polymerases are likely to be driving sunlight-induced skin carcinogenesis in XPV patients.Here, we show that 3 TLS polymerases, pol, pol, and pol, are involved in TLS across CPD in XPV cells. Moreover, pol and pol, but not pol, also provide protection against UV cytotoxicity, independently of nucleotide excision repair (NER). Results Pol, pol, and pol Are Involved in T...

Translesion Synthesis of 8,5′-Cyclopurine-2′-deoxynucleosides by DNA Polymerases η, ι, and ζ

“…In keeping with our study, two-polymerase mechanisms, in which insertion by one (or more) specific TLS Pol(s) is followed by extension with another polymerase, has been suggested to play important roles in the replicative bypass of several other DNA lesions in mammalian cells. These included thymidine glycol, cis,syn-cyclobutane pyrimidine dimer, and pyrimidine(6 -4)pyrimidone photoproducts in mammalian cells (6,42,43,51,52). It would be interesting to unveil how different inserter and extender Pols as proposed here and in other studies (6,42,43,51,52) coordinate their actions during TLS across DNA damage in human cells.…”

“…This result may reflect a role for the residual Pol in TLS across S-cdG in cells. In this vein, it was suggested that residual TLS Pols might be sufficient for keeping the miscoding properties of some DNA lesions unchanged, even though sometimes lesion bypass efficiencies could be reduced by the siRNA knockdown of some specific TLS Pols (37,42,43).…”

“…Highly efficient depletion of specific TLS DNA polymerases was achieved using siRNAs that were previously validated (42,43). All siRNAs were purchased from Dharmacon: POLK…”

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1

“…Recently, it has been shown that three TLS polymerases (Poli, Polj and Polf) are involved in mutagenic TLS across CPDs and two of them (Polj and Polf) protect Polg-deficient cells against UV cytotoxicity (Yoon et al 2009;Ziv et al 2009). However, this alternative TLS pathway does not explain how stalled inactive Polg would be replaced by other TLS polymerase(s).…”

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

“…Plasmid model systems were instrumental in the study of DNA damage tolerance via TLS (e.g., refs. [21][22][23][24][25] and HDR (12,26); however, they do not obviate the need for studying TLS and HDR at the chromosomal level. To that end we developed a unique method, in which both TLS and HDR can be simultaneously analyzed at a single-nucleotide resolution in chromosomes of mammalian cells in culture.…”