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

Xiaoxiao Wang,Szu Hua Sharon Lin,Zhongtao Zhang,Marietta Y.W.T Lee,Amal A Rahmeh,Sufang Zhang,Fu Yue,Ernest Y.C Lee,Rong Zheng

doi:10.18632/oncotarget.7034

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.

Highlights

Genomic stability in all organisms begins with the process of DNA replication, which is performed by replicative DNA polymerases endowed with exquisite fidelity
We first determined that PDIP46 interacts with the intact polymerase δ (Pol δ) heterotetramer, to ascertain that this interaction is not restricted to the free p50 subunit used as the bait in the yeast two-hybrid screen
This was established by the use of GST-PDIP46 pull-down assays, which demonstrated that all four subunits of the Pol δ4 were pulled down (Figure 1A)

Summary

Introduction

Genomic stability in all organisms begins with the process of DNA replication, which is performed by replicative DNA polymerases endowed with exquisite fidelity. An understanding of the properties and regulation of the replicative DNA polymerases is of crucial significance in the context of the maintenance of genome stability. The mechanisms by which the high fidelity of DNA polymerases is achieved have been extensively studied [1]. Mutations that alter the properties of Pol δ and Pol ε are of direct significance as a potential of genomic instability [2, 3]. It is well recognized that defects in the proofreading abilities of DNA polymerases in a broad range of organisms play a crucial role in increased mutation rate and genomic instability [4]. There remains a great deal yet to be learned about mechanisms that could affect the properties of the replicative polymerases and contribute to cancer etiology

Methods

Results

Conclusion