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.
Highlights
Accurate and processive DNA synthesis by DNA polymerases (Pol) during chromosomal DNA replication is essential for lowering the rate of spontaneous mutations and suppressing carcinogenesis (Pavlov et al, 2006)
Yeast two-hybrid assay and native PAGE analysis were carried out to examine the potential oligomerization status of the p12 subunit (Fig 1). p12 orf was fused in frame with both GAL4 activation (AD) and GAL4-binding domains (BD)
By taking advantage of native PAGE analysis where proteins resolve based on their charge and hydrodynamic size, we found that p12 migrated at a similar position with Carbonic anhydrase (CA) which is a protein of 30 kD with pI 6.4
Summary
Accurate and processive DNA synthesis by DNA polymerases (Pol) during chromosomal DNA replication is essential for lowering the rate of spontaneous mutations and suppressing carcinogenesis (Pavlov et al, 2006). The mechanism of DNA replication in higher eukaryotes is yet to be deciphered; Polδ replicates both the leading and lagging strands of the SV40 virus genome (Waga et al, 1994; Stillman, 2008). Irrespective of their different roles in DNA replication, these DNA polymerases possess certain commonalities such as the multi-subunit composition and signature sequences of a B-family DNA polymerase in the largest catalytic subunits (Tahirov et al, 2009; Kunkel & Burgers, 2017)
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