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.
Ohashi
Eiji
E
Institute for Virus Research, Kyoto University, Japan.
Hanafusa
Tomo
T
Kamei
Keijiro
K
Song
Ihnyoung
I
Tomida
Junya
J
Hashimoto
Hiroshi
H
Vaziri
Cyrus
C
Ohmori
Haruo
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques; D015003:Yeasts; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D009713:Nucleotidyltransferases; C498613:Polk protein, mouse; D000099270:Y-Family DNA Polymerases; C496086:Rev1 protein, mouse