Identification of a novel REV1‐interacting motif necessary for DNA polymerase κ function

Abstract

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 (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1-interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. PCNA-binding motifs found in many proteins also contain FF with some conserved residues N-terminal to FF, as frequently represented by Q-x-x-(I,L,M)-x-x-F-F (x, any residue). In contrast, our results show that the critical FF in RIR motifs are not flanked by specific conserved residues. Instead, the consensus sequence for REV1-binding is denoted by x-F-F-y-y-y-y (y, any residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polκ 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 Polκ function in vivo.

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

“…As shown in Fig. 5B, Polk ) ⁄ ) cells showed mild sensitivity to UV irradiation, as previously reported (Ogi et al 2002;Ohashi et al 2009), and…”

Structural Basis of Rev1-mediated Assembly of a Quaternary Vertebrate Translesion Polymerase Complex Consisting of Rev1, Heterodimeric Polymerase (Pol) ζ, and Pol κ

“…3C). Because these two Pol residues are not conserved and their alanine substitutions do not affect the Rev1 CTD-Pol RIR binding affinity (17), their crystallographically observed polar interactions may not contribute significantly to the binding energy.…”

“…Rev1 interaction is required for the protective role of Pol against benzo[a]pyrene in mammalian cells, and it promotes Pol -mediated suppression of spontaneous mutations in human cells (17,18). Although the interactions between the Rev1 CTD and the RIRs of Y-family polymerases , , and are only found in vertebrates (15), the Rev1 CTD-Pol interaction is evolutionarily conserved from yeast to humans, highlighting the essential role of the Rev1-Pol interaction in translesion synthesis.…”

Molecular Chaperone Hsp90 Regulates REV1-Mediated Mutagenesis

“…Based on these findings, it was proposed that REV1 acts as a scaffold protein to coordinate TLS polymerase switching (10,14,28,43,58). To support this notion, recent studies have indicated that the REV1/Pol interaction is required for Pol function (41) and that the REV1/Pol interaction promotes targeting of REV1 to UV-damaged DNA (1).Among the Y-Pols, REV1 and Pol are conserved throughout the eukaryotes and have been extensively studied. Substantial evidence indicates that posttranslational control of Pol plays an important role in the regulation of its function.…”