Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.

DNA damage tolerance and mutagenesis are hallmarks and enabling characteristics of neoplastic cells that drive tumorigenesis and allow cancer cells to resist therapy. The ‘Y-family’ trans-lesion synthesis (TLS) DNA polymerases enable cells to replicate damaged genomes, thereby conferring DNA damage tolerance. Moreover, Y-family DNA polymerases are inherently error-prone and cause mutations. Therefore, TLS DNA polymerases are potential mediators of important tumorigenic phenotypes. The skin cancer-propensity syndrome xeroderma pigmentosum-variant (XPV) results from defects in the Y-family DNA Polymerase Pol eta (Polη) and compensatory deployment of alternative inappropriate DNA polymerases. However, the extent to which dysregulated TLS contributes to the underlying etiology of other human cancers is unclear. Here we consider the broad impact of TLS polymerases on tumorigenesis and cancer therapy. We survey the ways in which TLS DNA polymerases are pathologically altered in cancer. We summarize evidence that TLS polymerases shape cancer genomes, and review studies implicating dysregulated TLS as a driver of carcinogenesis. Because many cancer treatment regimens comprise DNA-damaging agents, pharmacological inhibition of TLS is an attractive strategy for sensitizing tumors to genotoxic therapies. Therefore, we discuss the pharmacological tractability of the TLS pathway and summarize recent progress on development of TLS inhibitors for therapeutic purposes.

Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18−/− and Polκ−/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.