6. Regulation of Y-family translesion synthesis (TLS) DNA polymerases by RAD18

The recruitment of the error-prone Y-Family Translesion Synthesis (TLS) DNA polymerases (Pol , Pol , Pol , and REV1) to damaged chromatin is partly dependent on their association with Lysine 164 (K164)mono-ubiquitylated PCNA. RAD18 is the major PCNA K164-directed E3 ubiquitin ligase in eukaryotic cells and therefore plays potentially important roles in TLS and mutagenesis. Accordingly, there is considerable interest in elucidating the regulation of RAD18. Many molecular mechanisms that contribute to DNA damage-induced RAD18 activation have been identified. In S-phase, a protein complex containing RAD18 and Pol is redistributed to the vicinity of stalled replication forks, likely due to direct association of RAD18 with RPA-coated ssDNA, a substrate generated by uncoupling of replicative DNA polymerase and helicase activities induced by certain genotoxic injuries that leads to replication fork stalling. Correspondence/Reprint request: Dr. Cyrus Vaziri, Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA. E-mail: [email protected] Cyrus Vaziri et al. 92 Pol then facilitates the interaction of RAD18 with PCNA and promotes K164 mono-ubiquitylation. Pol mediates RAD18-PCNA interactions via a scaffold function that is fully separable from its DNA polymerase catalytic activity. Formation of the RAD18-Pol complex is partly dependent on S-phase checkpoint signaling and on RAD18 phosphorylation by DBF4-Dependent Kinase (DDK) and c-Jun N-terminal Kinase (JNK). Thus RAD18-dependent TLS is coordinated with DNA replication and other elements of the DNA damage response via multiple protein kinase signaling cascades. In addition to its S-phase functions, RAD18 can also be targeted to RPA-coated ssDNA generated during Nucleotide Excision Repair (NER) and Single-Stranded Break (SSB) repair in non-replicating cells. Therefore, RAD18-mediated PCNA mono-ubiquitylation and Y-family TLS polymerases also contribute to repair synthesis reactions independently of DNA replication. In addition to its key roles in TLS, RAD18 contributes to activation of several other genome maintenance mechanisms including the error-free Template Switch (TS) pathway (involving PCNA poly-ubiquitylation), Fanconi Anemia (FA) pathway activation, Homologous Recombination (HR), and Double-Stranded Break (DSB) processing via 53BP1. Thus RAD18 may provide a central nexus for crosstalk between multiple DNA damage tolerance and DNA repair mechanisms. Roles of Rad18 in vivo are not fully understood, yet Rad18 is required for the efficient repair of a small subset of meiotic DSB and long-term maintenance of germ cells in the mouse testes. The potential contribution of RAD18-mediated genome maintenance to tumorigenesis will also be considered in this chapter.