Abstract
DNA polymerase δ (Polδ) is a highly processive essential replicative DNA polymerase. In humans, the Polδ holoenzyme consists of p125, p50, p68 and p12 subunits and recently, we showed that the p12 subunit exists as a dimer. Extensive biochemical studies suggest that all the subunits of Polδ interact with the processivity factor proliferating cell nuclear antigen (PCNA) to carry out a pivotal role in genomic DNA replication. While PCNA-interacting protein motif (PIP) motifs in p68, p50 and p12 have been mapped, same in p125, the catalytic subunit of the holoenzyme, remains elusive. Therefore, in the present study by using multiple approaches we have conclusively mapped a non-canonical PIP motif from residues 999VGGLLAFA1008 in p125, which binds to the inter-domain-connecting loop (IDCL) of PCNA with high affinity. Collectively, including previous studies, we conclude that similar to Saccharomyces cerevisiae Polδ, each of the human Polδ subunits possesses motif to interact with PCNA and significantly contributes toward the processive nature of this replicative DNA polymerase.
Highlights
Eukaryotic DNA replication requires the concerted action of several enzymes and accessory factors [1,2,3]
Since a peptide derived from p21 inhibits p50 binding to proliferating cell nuclear antigen (PCNA), it suggested that p50 PCNA-interacting protein motif (PIP) motif has a weaker affinity toward the inter-domain-connecting loop (IDCL) of PCNA than that of p21
We have shown that p12 is a dimer in hPolδ, and the dimerization in the N-terminal RKR-motif induces its interaction with PCNA via PIP motif located at the C-terminal domain [17]
Summary
Eukaryotic DNA replication requires the concerted action of several enzymes and accessory factors [1,2,3]. While helicases and DNA polymerases are the key enzyme components; replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) are the integral structural components of the DNA replication machinery. These genes are essential for cell survival. Bacteria and viruses use the same processive DNA polymerase for both lagging and leading strands [4]. Extensive genetic studies mostly in yeast suggest that while DNA polymerase δ (Polδ) plays a key role in lagging strand synthesis, Polε plays a important role in leading strand synthesis [6]. It is important to understand the mechanism underlying processive DNA synthesis by Polδ and decipher precise contribution by its subunits in such an activity
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