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

Abstract

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 Euryarchaeota whose genome is sequenced, Thermococcus 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, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Dpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

Thermococcus kodakarensis DNA replication

“…It was found that the two proteins form homotrimers in solution with similar three-dimensional structures [57]. However, although both PCNA1 and PCNA2 stimulate the activity of PolB [57] and Fen1 (flap endonuclease 1) (Z. Li and Z. Kelman, unpublished work), PCNA1 is sufficient for cell viability ( [58], and T. Santangelo and Z. Kelman, unpublished work).…”

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

“…Indeed, one of the two PCNA proteins in T. kodakarensis bound to Hef at IDR. The bound PCNA was PCNA1, which is essential for the viability of T. kodakarensis and is probably a regular member of the replisome (35). PCNA1 in the progressing replisome can stimulate recruitment of Hef when the replication fork stalls.…”

“…On the other hand, the gene for PCNA2 can be knocked out and the physiological function of PCNA2 has not been elucidated. Furthermore, many more molecules of PCNA1 exist as compared with PCNA2 (35). We analyzed only PCNA1 in this study, and will see the relationships between PCNA2 and Hef in our further works.…”

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein

“…Genes encoding for PCNA are present in all archaeal genomes and while the genomes of most euryarchaeota contain a single gene encoding PCNA, the genome of Thermococcus kodakarensis contains two genes encoding PCNA homologues (TK0535 and TK0582, encoding PCNA1 and PCNA2, respectively) (20–22). It was found that both proteins form homotrimeric structures with characteristics similar to those of other archaeal and eukaryal PCNA proteins (20,23).…”

“…It was found that both proteins form homotrimeric structures with characteristics similar to those of other archaeal and eukaryal PCNA proteins (20,23). Both proteins were also shown to support processive DNA synthesis by DNA polymerase B (PolB) and polymerase D (PolD) (20–22). …”