Abstract
Family B DNA polymerases comprise polymerase and 3′ −>5′ exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein–protein and protein–DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a “brake” that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode.
Highlights
Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein that encircles DNA and plays numerous roles in nucleic-acid metabolism
Pyrococcus furiosus DNA polymerase BI (PfuPolB) is tightly anchored on PCNA by the PCNA interacting protein (PIP) motif, and rotates from the polymerase to exonuclease mode with the PIP motif as a pivot center (Fig. 1A, bottom)
Since the atomic resolution structures of the Pol/PCNA/DNA complex in either the polymerase or exonuclease mode have not been solved, the basic amino acid cluster ‘Site A’ focused on in this study was discovered by analyses of the structural model of the ternary complex
Summary
Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein that encircles DNA and plays numerous roles in nucleic-acid metabolism. DNA replication in Archaea and Eukarya is considered to be performed mainly by family B DNA polymerases, composed of the polymerase and exonuclease domains responsible for the DNA synthesizing and editing (proofreading) reactions, respectively, the polB gene can be deleted from some archaeal genomes and PolD probably functions in replication in those cases[13,14]. These replication enzymes exhibit full activity when complexed with PCNA. The replicating complex structure of a family B DNA polymerase revealed that the substrate DNA interacted solely with the polymerase domain, while the substrate DNA in the editing complex interacted exclusively with the exonuclease domain[15,16,17]
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