Structural basis for novel interactions between human translesion synthesis polymerases and proliferating cell nuclear antigen.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Poleta, Poliota, and Polkappa. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Poldelta, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Poleta, Poliota, and Polkappa have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Poliota adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

Identification of a novel REV1-interacting motif necessary for DNA polymerase kappa function.

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Poleta, Poliota, Polkappa and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Poleta, Poliota, and Polkappa and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polkappa. We have identified novel REV1-interacting regions (RIRs) present in Polkappa, Poliota and Poleta. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polkappa mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk-null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polkappa function in vivo.