Structure of a Sliding Clamp on DNA

Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10021, USA.
Cell (Impact Factor: 32.24). 02/2008; 132(1):43-54. DOI: 10.1016/j.cell.2007.11.045
Source: PubMed


The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.

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    • "In fact, depletion of both CBP and p300 resulted in an accumulation of chromatin-bound PCNA to DNA damage sites which might delay the coordinated hand-off of intermediates during DNA repair (55). Since acetylation will abolish the charge of lysines contacting DNA (55,67), further structural studies are required to explain the impact of this PCNA modification on DNA synthesis. "
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    ABSTRACT: The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku533 · 9.11 Impact Factor
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    • "Protein-protein interactions are central to such regulation within the E. coli replisome and characterisation of these interactions is critical to our understanding of these complex, dynamic processes [1]. Processivity factors play an integral role involving both protein-protein and protein-DNA interactions in regulation of events at the replication fork [2,3]. "
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    ABSTRACT: Strict regulation of replisome components is essential to ensure the accurate transmission of the genome to the next generation. The sliding clamp processivity factors play a central role in this regulation, interacting with both DNA polymerases and multiple DNA processing and repair proteins. Clamp binding partners share a common peptide binding motif, the nature of which is essentially conserved from phage through to humans. Given the degree of conservation of these motifs, much research effort has focussed on understanding how the temporal and spatial regulation of multiple clamp binding partners is managed. The bacterial sliding clamps have come under scrutiny as potential targets for rational drug design and comprehensive understanding of the structural basis of their interactions is crucial for success. In this study we describe the crystal structure of a complex of the E. coli beta-clamp with a 12-mer peptide from the UmuC protein. UmuC is the catalytic subunit of the translesion DNA polymerase, Pol V (UmuD'2C). Due to its potentially mutagenic action, Pol V is tightly regulated in the cell to limit access to the replication fork. Atypically for the translesion polymerases, both bacterial and eukaryotic, Pol V is heterotrimeric and its beta-clamp binding motif (357 QLNLF 361) is internal to the protein, rather than at the more usual C-terminal position. Our structure shows that the UmuC peptide follows the overall disposition of previously characterised structures with respect to the highly conserved glutamine residue. Despite good agreement with the consensus beta-clamp binding motif, distinct variation is shown within the hydrophobic binding pocket. While UmuC Leu-360 interacts as noted in other structures, Phe-361 does not penetrate the pocket at all, sitting above the surface. Although the beta-clamp binding motif of UmuC conforms to the consensus sequence, variation in its mode of clamp binding is observed compared to related structures, presumably dictated by the proximal aspartate residues that act as linker to the poorly characterised, unique C-terminal domain of UmuC. Additionally, interactions between Asn-359 of UmuC and Arg-152 on the clamp surface may compensate for the reduced interaction of Phe-361.
    BMC Structural Biology 07/2013; 13(1):12. DOI:10.1186/1472-6807-13-12 · 1.18 Impact Factor
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    • "The DNA-free protein structures are remarkably similar and reveal an open state that closes on binding primer-template DNA (discussed in 25). In addition, the crystal structures of an E. coli β2:dsDNA complex (23), ε186 (12) and the ε186:HOT complex (13,14) are known, as well as the NMR structure of the ε186:θ complex (16). "
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    ABSTRACT: A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.
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