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  • Human DNA polymerase delta is a pentameric holoenzyme with a dimeric p12 subunit.

Human DNA polymerase delta is a pentameric holoenzyme with a dimeric p12 subunit.

Life science alliance (2019-03-20)
Prashant Khandagale, Doureradjou Peroumal, Kodavati Manohar, Narottam Acharya
ABSTRACT

Human DNA polymerase delta (Polδ), a holoenzyme consisting of p125, p50, p68, and p12 subunits, plays an essential role in DNA replication, repair, and recombination. Herein, using multiple physicochemical and cellular approaches, we found that the p12 protein forms a dimer in solution. In vitro reconstitution and pull down of cellular Polδ by tagged p12 substantiate the pentameric nature of this critical holoenzyme. Furthermore, a consensus proliferating nuclear antigen (PCNA) interaction protein motif at the extreme carboxyl-terminal tail and a homodimerization domain at the amino terminus of the p12 subunit were identified. Mutational analyses of these motifs in p12 suggest that dimerization facilitates p12 binding to the interdomain connecting loop of PCNA. In addition, we observed that oligomerization of the smallest subunit of Polδ is evolutionarily conserved as Cdm1 of Schizosaccharomyces pombe also dimerizes. Thus, we suggest that human Polδ is a pentameric complex with a dimeric p12 subunit, and discuss implications of p12 dimerization in enzyme architecture and PCNA interaction during DNA replication.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-POLD4 antibody produced in mouse, clone 2B11, ascites fluid
Sigma-Aldrich
Anti-PCNA, IgG fraction of antiserum
Sigma-Aldrich
Anti-DNA polymerase δ p125 antibody, Rat monoclonal, clone PDG 5G1, purified from hybridoma cell culture
Sigma-Aldrich
Anti-DNA polymerase δ p50 antibody, Rat monoclonal, clone PDK 7B4, purified from hybridoma cell culture
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, affinity isolated antibody, buffered aqueous solution