Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen ( PCNA ) is the sliding clamp. The ring‐shaped PCNA encircles double‐stranded DNA within its central hole and tethers other proteins on DNA . The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the E uryarchaeota whose genome is sequenced, T hermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T . kodakarensis genome, and the gene products, PCNA 1 and PCNA 2, were characterized. PCNA 1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T . kodakarensis in vitro . PCNA 2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA 2, whereas gene disruption for PCNA 1 was not possible, suggesting that PCNA 1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation ( UV ), methyl methanesulfonate ( MMS ) and mitomycin C ( MMC ) were indistinguishable from those of the wild‐type strain.