Abstract
PDIP46 (poldip3) is a 46 kDa protein which interacts with human DNA polymerase δ (Pol δ) via the p50 subunit, and whose biochemical effects on Pol δ are unknown. The objectives of this study were to characterize the effects of PDIP46 on Pol δ activity. The interaction sites between the p50 subunit of Pol δ and PDIP46 were mapped. PDIP46 was also found to interact with PCNA, which it does via multiple copies of a novel PCNA binding motif (APIM ‐ AlkB homologue 2 PCNA‐Interacting Motif). Both the p50 and PCNA interaction regions are located in the N‐terminus. PDIP46 potently activates Pol δ activity (ca. 10‐fold with an apparent KD of ca. 34 nM) on singly primed 7 kb ssM13 DNA in an assay which requires highly processive synthesis. Further dissection of the effects of PDIP46 using model oligonucleotide templates showed that it stimulated primer extension and strand displacement reactions in the presence of PCNA. PDIP46 also stimulated primer extension in the absence of PCNA, indicating that it has the ability to exert a direct effect on Pol δ. PDIP46 was associated with Pol δ on DNA as shown by chromatin immunoprecipitation using antibody against the Pol δ p125 subunit. These in vitro studies show that PDIP46 has the biochemical attributes to function as a novel accessory protein for Pol δ that accelerates its activity, and thus is a candidate for a role as a cellular regulator of Pol δ.
Published Version
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