Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

The Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, P. O. Box 228, New York, NY 10065, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2008; 105(32):11116-21. DOI: 10.1073/pnas.0804754105
Source: PubMed

ABSTRACT DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.

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Available from: Seung-Sup Kim, Sep 26, 2015
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    • "Because transposases are frequently toxic and insoluble when overexpressed, we exploited the fact that β-binding motifs are often located on highly flexible, peptide-like, structures at the C-terminus of the protein (Dalrymple et al. 2001; Bunting et al. 2003; López de Saro et al. 2003). Further, unlike most protein–protein interactions, which implicate relatively large surface areas, interactions with β are mostly circumscribed to the motif binding to a hydrophobic pocket on β (Georgescu et al. 2008). We synthesized N-biotinylated peptides (20 aa) derived from sequences of transposases containing putative β-binding motifs (fig. "
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    ABSTRACT: Insertion Sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to 9 different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. While we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, since in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phyllogenetically distant species.
    Genome Biology and Evolution 03/2014; 6(3). DOI:10.1093/gbe/evu052 · 4.23 Impact Factor
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    • "The Pol III C-terminal peptide has one aspartate residue at the extreme C-terminus. In contrast to that seen in UmuC, for which no density was observed, the terminal residue of the Pol III peptide was well ordered, presumably a reflection of the intimate contact made between the clamp Arg-246 and the preceding phenylalanine in the peptide [14]. Presumably Asp-362 and Asp-363 do not form sufficiently stable contacts with the clamp surface for electron density to be observed. "
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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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    • "Most other clamp-binding proteins rely primarily on one tight interaction with the clamp. For example, most DNA polymerases [12,67,76,77] and the cell cycle regulator p21 [68] interact with the clamp primarily through a single, high-affinity binding site. The weak cooperative interactions between clamp loader and clamp are easily regulated to facilitate clamp loader ejection after loading the clamp onto DNA (see below). "
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    ABSTRACT: Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.
    BMC Biology 04/2012; 10(1):34. DOI:10.1186/1741-7007-10-34 · 7.98 Impact Factor
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