Identification of a novel REV1-interacting motif necessary for DNA polymerase κ 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 (Pol eta, Pol iota, Pol kappa and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Pol eta, Pol iota, and Pol kappa and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Pol kappa. We have identified novel REV1-interacting regions (RIRs) present in Pol kappa, Pol iota and Pol eta. 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 Pol kappa 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 Pol kappa function in vivo.