| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2006: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2005: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 2004: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Research Abstract |
We and other groups have shown that the three Y-family DNA polymerases Polη, Polι, and Polκ interact with a C-terminal region of REV 1, another member of the Y-family DNA polymerases. The same C-terminal region of hREV1 was also shown to interact with hREV7, a non-catalytic subunit of Polζ, which is another TLS DNA polymerase belonging to the B-family. This has suggested us that hREV1 plays a crucial role in cellular TLS events. Unlike for the PCNA binding, Polη, Polι, and Polκ did not seem to have an apparent consensus sequence for the hREV1-interaction. However, our further experiments revealed that short sequences less than 10 residues are sufficient for the hREV1-interaction in all of the three Pols. In addition, in all cases, two consecutive phenylalanine (FF) residues were found to be critical for the hREV1-interactions. No conserved amino acid residue is required for the N-terminal side of the FF motif, but the presence of several residues at its C-terminal side is essential while the sequence is not conserved. Using surface plasmon resonance method, we showed that short synthetic peptides of the sequence derived from the hREV1-binding sites of Polκ directly bind the C-terminal region of hREV1. Furthermore, such peptides did inhibit the binding between purified hREV1 and Polκ proteins.
To examine the biological relevance of the interaction between hREV1 and Polκ, we employed complementation assay using Polκ-deficient MEF (mouse embryonic fibroblast) cells, which exhibit increased sensitivity to BPDE (benzo[a[pyrene dihydroxydiol epoxide, the ultimate carcinogen of benzo[a[pyrene] and UV-irradiation. We observed that the sensitivities to BPDE or UV were recovered by transient expression of the wild-type of Polκ (in a fusion with GFP), but not by that of the FF567-578AA mutant deficient for the hREV1-binding.
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