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

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.

Identification of PCNA interacting protein motifs in human DNA polymerase delta

“…2 A II). The crystal structure of the PIP box of p68 (456QVSITGFF463) and modeled the structure of PIP of p12 (98QCSLWHLY105) exhibit remarkable structural conservation and form 310 helices [16]. However, according to the p50-p68 co-crystal structure, the PIP motif of p50 (58QMRPFL65) is located in the 2 helix of the OB-fold domain and is not involved in an interaction with p68 subunit [24].…”

“…To achieve higher processivity in vitro, all three PIP boxes are required; however for the cellular function of ScPolδ, along with PIP of ScPol32, one more PIP box from either Pol3 or Pol31 subunit are essential. Interestingly, human Pol consists of the catalytic large subunit p125 (PolD1) and accessory subunits p50 (PolD2), p68 (PolD3), and p12 (PolD4) [16,17]. Recently, we have shown that p12 exists as a dimer both in solution as well as in the holoenzyme [16].…”

“…Interestingly, human Pol consists of the catalytic large subunit p125 (PolD1) and accessory subunits p50 (PolD2), p68 (PolD3), and p12 (PolD4) [16,17]. Recently, we have shown that p12 exists as a dimer both in solution as well as in the holoenzyme [16]. In several studies, biochemical interaction between each subunit of hPol and PCNA has been demonstrated [17][18][19].…”

“…We showed that the oligomerization of p12 at the amino-terminal domain facilitates its interaction with PCNA at the carboxyl-terminal domain. Both the oligomerization and the PIP motifs of p12 have been mapped [16]. The co-crystal structure of a peptide containing the PIP motif of p68 with PCNA has been solved [20].…”

Virulence and pathogenicity of aCandida albicansmutant with reduced filamentation

“…Accurate and processive DNA synthesis by DNA polymerases (Pol) during replication, repair, and recombination is essential for lowering the rate of spontaneous or damage-induced genome instability (Khandagale, Peroumal, Manohar, & Acharya, 2019;Prakash, Johnson, & Prakash, 2005). As C. albicans exhibits considerable genotypic variation both in vitro and in clinical isolates due to genetic alterations (Ahmad et al, 2008;Ciudad, Hickman, Bellido, Berman, & Larriba, 2016;Rustchenko, 2007), DNA polymerases (Pol) could play a crucial role in fungal pathogenesis.…”