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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.

SUBMITTER: Nishida H

PROVIDER: S-EPMC2791594 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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

Nishida Hirokazu H Mayanagi Kouta K Kiyonari Shinichi S Sato Yuichi Y Oyama Takuji T Ishino Yoshizumi Y Morikawa Kosuke K

Proceedings of the National Academy of Sciences of the United States of America 20091123 49

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 inter ...[more]

PMID: 19934045

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Project description:Replicative DNA polymerases (Pols) frequently possess two distinct DNA processing activities: DNA synthesis (polymerization) and proofreading (3'-5' exonuclease activity). The polymerase and exonuclease reactions are performed alternately and are spatially separated in different protein domains. Thus, the growing DNA primer terminus has to undergo dynamic conformational switching between two distinct functional sites on the polymerase. Furthermore, the transition from polymerization (pol) mode to exonuclease (exo) mode must occur in the context of a DNA Pol holoenzyme, wherein the polymerase is physically associated with processivity factor proliferating cell nuclear antigen (PCNA) and primer-template DNA. The mechanism of this conformational switching and the role that PCNA plays in it have remained obscure, largely due to the dynamic nature of ternary Pol/PCNA/DNA assemblies. Here, we present computational models of ternary assemblies for archaeal polymerase PolB. We have combined all available structural information for the binary complexes with electron microscopy data and have refined atomistic models for ternary PolB/PCNA/DNA assemblies in pol and exo modes using molecular dynamics simulations. In addition to the canonical PIP-box/interdomain connector loop (IDCL) interface of PolB with PCNA, contact analysis of the simulation trajectories revealed new secondary binding interfaces, distinct between the pol and exo states. Using targeted molecular dynamics, we explored the conformational transition from pol to exo mode. We identified a hinge region between the thumb and palm domains of PolB that is critical for conformational switching. With the thumb domain anchored onto the PCNA surface, the neighboring palm domain executed rotational motion around the hinge, bringing the core of PolB down toward PCNA to form a new interface with the clamp. A helix from PolB containing a patch of arginine residues was involved in the binding, locking the complex in the exo mode conformation. Together, these results provide a structural view of how the transition between the pol and exo states of PolB is coordinated through PCNA to achieve efficient proofreading.

Dataset Information

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

Publications

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

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