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

Abstract

DNA polymerase g (Polg), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polg interacts with REV1, and REV1 interacts with other TLS polymerases including Poli, Polj and Polf, Polg may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polg activity but also by dysfunction of network between Polg and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polg is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polg-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polg. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polg and REV1. However, expression of the inactive mutant Polg was not able to suppress the incidence of UV-induced mutation observed in Polg-deficient cells. We propose the model that REV1 and Polj are involved in DNA damage tolerance via Polg-REV1 interaction when Polg fails to bypass its cognate substrates.

A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

“…We show that the Polη–Rad18 interaction provides the basis for coupling PCNA monoubiquitination with DNA damage-inducible checkpoint pathways mediated by p53 and Chk1. Our results also provide a potential explanation for numerous reports that Polη confers tolerance of non-cognate lesions (33,34) and that catalytically inactive Polη can partially rescue the DNA damage-sensitivity phenotypes of XPV cells (35,36). Moreover, because some XPV cells express a catalytically inactive Polη that retains the ability to promote PCNA monoubiquitination, our results also indicate a new molecular mechanism for the mutagenesis and cancer propensity of XPV patients.…”

“…For example, XPV cells are hypersensitive to BPDE and other genotoxins whose DNA lesions are not bypassed by Polη (33,34); clearly, a polymerase-independent function of Polη that promotes PCNA monoubiquitination and activation of Polκ (the TLS polymerase that mediates bypass of BPDE adducts) explains the BPDE sensitivity of XPV cells. In other studies, catalytically dead Polη mutants conferred DNA damage tolerance (36,59) and mutagenesis (35,36). Because PCNA monoubiquitination at K164 is necessary for tolerance of UV and other genotoxins (15–17), restoration of UV survival by catalytically dead Polη (36) is explained by its scaffold function that promotes PCNA monoubiquitination, thus recruiting other TLS polymerases that facilitate tolerance, albeit at a cost of increased mutagenesis.…”

“…In other studies, catalytically dead Polη mutants conferred DNA damage tolerance (36,59) and mutagenesis (35,36). Because PCNA monoubiquitination at K164 is necessary for tolerance of UV and other genotoxins (15–17), restoration of UV survival by catalytically dead Polη (36) is explained by its scaffold function that promotes PCNA monoubiquitination, thus recruiting other TLS polymerases that facilitate tolerance, albeit at a cost of increased mutagenesis.…”

NMR Structure and Dynamics of the C-Terminal Domain from Human Rev1 and Its Complex with Rev1 Interacting Region of DNA Polymerase η

“…Example of cognate lesions include thymine-thymine cyclobutane pyrimidine dimers for polη, N 2 -BPDE-dG lesions for polκ, and εdA for polι 1,5 . Polymerase exchange mediated by the Rev1 C-terminal domain could also help exchange a non functional TLS polymerase for an alternative TLS polymerase 81 . Evidence has been reported indicating that, in higher eukaryotes, a component of TLS can also proceed in the absence of PCNA monoubiquitination 82 .…”

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells

“…Rev1 −/− MEFs (32), Rev3l −/− Trp53 −/− MEFs (33) and Polh −/− Poli −/− Polk −/− TKO MEFs (24, 34) have been described. The genomic reconfirmation of these knockouts is presented in Supplementary Fig.…”