the function of the motifs. The crystal structure of P. furiosus Pol B complexed with a monomeric PCNA mutant was determined, and a convincing model of the polymerase-PCNA ring interaction was constructed. This study revealed that a novel interaction is formed between a stretched loop of PCNA and the thumb domain of Pol B, in addition to the authentic PIP box. comparison of the model structure with the previously reported structures of a family B DNA polymerase from RB69 phage, complexed with DNA, suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. putative mechanism for the fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site enhanced the exonuclease activity in the presence of PCNA. Furthermore, the three-dimensional structure of the DNA polymerase-PCNA-DNA ternary complex was analyzed by electron microscopic (EM) single particle analysis. structural view revealed the entire domain configuration of the trimeric ring of PCNA and DNA polymerase, including the protein-protein or protein-DNA contacts. This architecture provides clearer insights into the switching mechanism between the editing and synthesis modes.
Figure 5. Mechanisms of processive DNA synthesis
In contrast to most euryarchaeal organisms, which have a single PCNA homolog forming a homotrimeric ring structure, the majority of crenarchaea have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. It is especially interesting that the three PCNAs, PCNA1, PCNA2, and PCNA3, specifically bind PCNA binding proteins, including DNA polymerases, DNA ligases, and FEN - 1 endonuclease. structural studies of the heterologous PCNA from S. solfataricus revealed that the interaction modes between the subunits are conserved with those of the homotrimeric PCNAs. T. kodakarensis is the only euryarchaeal species that has two genes encoding PCNA homologs on the genome. These two genes from the T. kodakarensis genome, and the highly purified gene products, PCNA1 and PCNA2, were characterized. stimulated the DNA synthesis reactions of the two DNA polymerases, Pol B and Pol D, from T. kodakarensis in vitro . PCNA2 however only had an effect on Pol B. The T. kodakarensis strain with pcna2 disruption was isolated, whereas gene disruption for pcna1 was not possible. These results suggested that PCNA1 is essential for DNA replication, and PCNA2 may play a different role in T. kodakarensis cells. sensitivities of the? pcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable to those of the wild type strain. Both PCNA1 and PCNA2 form a stable ring structure and work as a processivity factor for T. kodakarensis Pol B in vitro . The crystal structures of the two PCNAs revealed the different interactions at the subunit-subunit interfaces. the other hand, the archaeal RFC cons...
