Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis

Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the ␤-sheets that span the intermolecular interfaces, with smaller but similar contributions from ␤-sheets that span the intramolecular interfaces within each subunit. With the integrity of these ␤-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.clamp loader ͉ DNA replication ͉ DNA sliding clamps ͉ ␤-sheet distortion ͉ conformational transitions

supporting

confidence: 82%

Section: Transient Conformations Of Pcna Allow Extensive Engagement Withmentioning

confidence: 99%

Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

Kazmirski

Zhao

Bowman

et al. 2005

“…In the 3D structure of the DNA ligase-PCNA-DNA complex obtained by EM analysis (18), the dsDNA was tilted by 16°against the PCNA axis and had only one contact with the DNA. Meanwhile, in the clamp loading complex composed of primed DNA, PCNA, and replication factor C (RFC), the dsDNA was almost parallel to the PCNA axis, and no significant interactions were observed between DNA and PCNA (17). These results suggest that the configurations of PCNA are variable relative to the clamped dsDNA, depending upon the protein factors within the complexes.…”

Section: Resultsmentioning

confidence: 83%

“…Analyses of these structures suggested that the complex must undergo a considerable conformational change during the switch between the polymerizing and editing modes. Our studies, using single particle electron microscopy (EM), revealed that structural views of complexes at medium resolution can provide valuable information about the molecular mechanisms of the clamp loading and the DNA ligation reaction on PCNA (17,18). Here, we report direct visualization of the 3D structure of the Pyrococcus furiosus (Pfu) PolB-PCNA-DNA complex examined by single particle electron microscopy.…”

mentioning

confidence: 99%

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

Mayanagi

Kiyonari

Nishida

et al. 2011

Self Cite

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the threedimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNAinteracting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.fidelity control | protein-DNA complex | replication fork | single particle analysis | structural bioinformatics

“…EM analysis of PCNA/Pol-B/DNA and PCNA/ligase/DNA assemblies suggested that DNA was tilted by 13°and 16°, respectively (42,43). By contrast, in the replication factor C/ PCNA/DNA ternary assembly, the DNA axis was found to be nearly perpendicular to the PCNA ring (44). This versatile mode of association between the sliding clamp and DNA appears to be a universal feature observed in bacterial, archaeal, and eukaryotic clamps, and may allow for distinct functionalities with specific protein partners.…”

Section: Structural Determinants Of Flexibility For the Ternary Assemmentioning

confidence: 99%

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

Querol-Audí

Cheng

et al. 2012

Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.F lap endonuclease 1 (FEN1) belongs to a class of essential nucleases (the FEN1 5′ nuclease superfamily) present in all domains of life (1). FEN1 catalyzes the endonucleolytic cleavage of bifurcated DNA or RNA structures known as 5′ flaps. These 5′ flaps are generated during lagging strand DNA synthesis or during long-patch base excision repair. The FEN1 substrates are in fact double-flap DNA (dfDNA) with DNA on the opposite side of the 5′ flap, forming a single nucleotide 3′ flap when bound to the enzyme (2, 3). By removing the 5′ ssDNA or RNA flap from such substrates, FEN1 produces a single nicked product that could be sealed by the subsequent action of a DNA ligase (4). Consistent with its crucial role in DNA replication and repair, FEN1 is highly expressed in all proliferative tissues, and its activity is key for the maintenance of genomic integrity (5). FEN1 has been identified as a cancer susceptibility gene, and mutations in it have been linked to a number of genetic diseases, such as EM map myotonic dystrophy, Huntington disease, several ataxias, fragile X syndrome, and cancer (6-10).The nuclease activity of FEN1 can be stimulated by association with processivity clamps such as proliferating cell nuclear antigen (PCNA), which encircle DNA at sites of replication and repair (11)(12)(13). PCNA is a recognized master coordinator of cellular responses to DNA damage and interacts with numerous DNA repair and cell-cycle control proteins. In this capacity, PCNA serves not only as a mobile platform for the attachment of these proteins to DNA but, importantly, plays an active role in the recru...