Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex

Abstract

Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with the cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction, with unprecedented configurations of the two molecules. Electron microscopic analyses indicated that this complex structure exists in solution. Our structural study revealed that an interaction occurs between a stretched loop of PCNA and the PfuPol Thumb domain, in addition to the authentic PCNA-polymerase recognition site (PIP box). Comparisons of the present structure with the previously reported structures of polymerases complexed with DNA, suggested that the second interaction plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. This putative mechanism for fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site caused enhancements in the exonuclease activity in the presence of PCNA.DNA clamp ͉ DNA replication ͉ electron microscopy ͉ fidelity control ͉ protein crystallography D NA replication is a highly coordinated process, in which DNA polymerase and the DNA sliding clamp play major roles in ensuring accurate genome duplication. Most replicative DNA polymerases consist of two independent functional components: polymerase and exonuclease moieties, which execute DNA synthesis and editing, respectively (1, 2). Since these two active sites are apart from each other, the DNA substrate cannot access both sites at the same time. These two reactions are performed alternately, and thus require regulation by temporally different switching, which was previously proposed from a comparison of the crystal structures of RB69 DNA polymerases complexed with dsDNA in the polymerase and exonuclease modes (3, 4).The DNA clamp, which forms a dimer or trimer, depending on the domain of life, retains DNA polymerase on the template DNA strand for processive DNA synthesis. DNA clamps, which are called PCNA in Archaea and Eukarya, and the ␤ subunit in Bacteria, form similar architectures with a central hole. Thus, they encircle the substrate DNA, thereby enabling consecutive DNA synthesis (5).PCNA-interacting proteins contain a small conserved motif, the PIP (PCNA-interacting protein) box, which binds to a common site on PCNA (6, 7). A model building study between the RB69 DNA polymerase-editing complex and PCNA revealed the consistent configuration of each component (3). The structure of the complex between the ''little finger'' (LF) domain of the translesional Y-family DNA polymerase Pol IV and the ␤-clamp from Escherichia coli showed that the bound polymerase domain lies at the outer rim of the clamp ring (8).We reported previously that the PCNA from the hyperthermophilic archaeon, Pyrococcus furiosus, bound to the PIP box of the family B D...

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

“…We previously proposed a model where E171, which is close to the authentic PIP-box contact, is detached from R706 of PfuPolB upon the transition from the polymerizing mode to the editing mode (10). This switching model is essentially consistent with our current model of the editing mode of PolB.…”

“…2G). In the previously reported PfuPolB-PCNA crystal structure, this turn was referred to as a "switching hook," and E171 was proposed to play a key role in switching between the editing and polymerizing modes (10). This switching hook was considered to function in the vicinity of the PIP-box contact site on the same PCNA subunit.…”

“…Several crystal structures of PolB have been determined, and they share essentially the same architecture, consisting of five domains: finger, palm, thumb (the three polymerase domains), N-term, and 3 0 → 5 0 exonuclease domains (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The exonuclease domain, which is responsible for the 3 0 → 5 0 editing of incorrect synthesis by the polymerizing activity, ensures the high fidelity of DNA replication (11,12).…”

“…Although the entire structure of the DNA polymerase-PCNA-DNA complex has not been reported yet, ternary complex models were proposed for both the polymerizing and editing modes of a bacteriophage system, based on the two crystal structures of the DNA polymerase-DNA complexes in each mode (1,7), and for that of the sliding clamp complexed with the clamp-binding fragment of the DNA polymerase, by using proteins from bacteriophage RB69 (1). Recently, the crystal structure of an archaeal full-length DNA polymerase complexed with a monomeric PCNA allowed us to construct a reasonable model of the ternary complex with DNA, thus providing clearer insights into the switching mechanism between the polymerizing and editing modes (10). Analyses of these structures suggested that the complex must undergo a considerable conformational change during the switch between the polymerizing and editing modes.…”

Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

“…1A) that bind to the IDCL of their respective PCNAs. Many studies suggest the importance of the PIP box in PCNA‐mediated interactions 6, 41, 42; however, our knowledge regarding specific surface interaction between PCNA and its protein partners is still incomplete. Herein, we demonstrate that in the case of DNA ligase, the PCNA interaction is partially independent of the PIP box.…”