PDIP46 (DNA polymerase δ interacting protein 46) is an activating factor for human DNA polymerase δ

Abstract

PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability.

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

“…The PCNA binding of PDIP46 is due to its possession of a cluster of 5 APIM motifs [56]. The APIM (AlkB homologue 2 PCNA-Interacting Motif) is a novel PCNA binding motif that was first identified in the human DNA repair enzyme oxidative demethylase ABH2 (AlkB homologue 2) [147].…”

“…This has been demonstrated by their co-immunoprecipitation and co-elution during affinity chromatography on immobilized anti-p125 monoclonal antibody. ChIP analysis with antibody against the p125 subunit showed that PDIP46 was present together with two components of the mammalian replisome [56]. These are Mcm2, a component of the CMG (Cdc45-MCM-GINS) helicase [151], and Ctf4, which associates with CMG [152,153].…”

“…10 fold) of Pol δ4 in the synthesis of the 7kb M13 DNA, with an apparent Kd of ca. 34 nM [56]. The mechanisms for this activation could be due to several causes.…”

“…These include an increase in processivity, possibly because PDIP46 may stabilize Pol δ binding to PCNA by a bridging interaction, as well as by a direct activation that involves alteration of the kinetic properties of Pol δ4. The effects of PDIP46 were examined on model oligonucleotide templates [127] in assays that examined primer extension and strand displacement in order to gain insights into its mechanism(s) of action [56]. In the absence of PCNA, Pol δ4 behaves in a distributive fashion, and PDIP46 clearly stimulates this activity.…”

“…In this model ( Figure 6) Pol δ3 is the default lagging strand polymerase. When regions of secondary structure which act as barriers to Pol δ3 are encountered [56], Pol δ4 is switched with Pol δ3, together with PDIP46 ( Figure 6). There are regions of varying template complexity in chromatin that include simple hairpins, microsatellite regions [159] that contain CFS (common fragile sites), and trinucleotide repeats [160].…”