Crystal Structure of Rat DNA Polymerase β: Evidence for a Common Polymerase Mechanism

Sawaya, M.R.; Pelletier, H.; Kumar, Arun; Wilson, Samuel H.; Kraut, Joseph

doi:10.1126/science.7516581

Cited by 474 publications

(455 citation statements)

References 48 publications

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“…We initially focused on the characterization of the complex formed between the XRCC1-NTD and the Pol β catalytic domain (Pol β CD), because it appears that the N-terminal Pol β lyase domain does not participate in this interaction, and because the position of this domain can be variable in the absence of a gapped DNA substrate (12). The interaction of XRCC1-NTD with the Pol β CD was first investigated using small-angle x-ray scattering (SAXS) ( Table S1).…”

Section: Resultsmentioning

confidence: 99%

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

Cuneo

London

2010

Proc. Natl. Acad. Sci. U.S.A.

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Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase β (Pol β) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol β have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol β, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.disulfide switch | DNA repair | scaffolding protein

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Section: Resultsmentioning

confidence: 99%

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

Cuneo

London

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Crystal structures have indicated that pol ␤ positions nucleotide substrates through interactions with all three components of the molecule: the base moiety pairs with the template base, the phosphate groups coordinate the catalytic Mg 2ϩ ions, and the ribose ring contacts the protein side chains and backbone (5,21). In crystal soaking experiments, Pelletier et al (23) found that AZT-TP could enter the nucleotide binding pocket, but that steric clash of the azide group with the protein backbone at residues 271-274 forced the nucleotide into a distorted conformation in which not only the sugar ring but the base and the phosphates are positioned incorrectly.…”

Section: Discussionmentioning

confidence: 99%

3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

Kosa

Sweasy

1999

Journal of Biological Chemistry

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We developed an in vivo selection to identify 3-azido-3-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase ␤ (pol ␤). The selection utilizes pol ␤'s ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol ␤ allows SC18-12 cells to grow, but they depend on pol ␤ activity, so inhibition of pol ␤ by AZT kills them. We screened a library of randomly mutated pol ␤ cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steadystate catalytic efficiency (K cat /K m ) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher K m (dTTP) and K cat (dTTP) values, implying that the incorporation reaction is altered. These are the first pol ␤ mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.Efficient and accurate synthesis of DNA, during replication and repair, is essential to the integrity of any genome. Template-directed synthesis requires that a polymerase select the appropriate deoxynucleotide triphosphate (dNTP) and exclude incorrect bases. The interaction between a polymerase and substrates must therefore be highly specific, yet flexible, in order to maintain sequence fidelity. The smallest of the eukaryotic enzymes that accomplish this reaction is DNA polymerase ␤ (pol ␤), 1 a mammalian polymerase which fills short gaps in DNA (1). pol ␤ has been implicated in base excision repair (2, 3) and meiosis (4). pol ␤ is highly suitable for structure-function studies of the molecular mechanism of DNA synthesis due to the availability of information on the polymerase-DNA-substrate ternary complex (5, 6).DNA synthesis by pol ␤ can be compromised by the nucleoside analog drug AZT, which closely resembles a normal substrate. AZT-triphosphate (AZT-TP) presents a normal thymine moiety which may form a Watson-Crick base pair with adenosine, but has a modified sugar ring that results in chain termination. pol ␤ incorporates AZT into DNA in vitro (7) implying that pol ␤ is not able to distinguish perfectly between the drug and the natural nucleotide substrate. This susceptibility of pol ␤ makes AZT resistance a useful probe of enzyme-substrate interactions involved in DNA synthesis.The molecular basis for polymerase substrate specificity, and specifically AZT discrimination, is not well understood. The clinical problem of AZT-resistant HIV has been attributed to mutations in HIV reverse transcriptase. However, the mutant reverse transcriptase enzymes have exhibited little or no change in AZT-TP incorporation...

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“…DNA polymerase ). Crystal structures of representative enzymes from the first four families have been determined, revealing a common overall architecture that has been likened to a human right hand, with fingers, thumb, and palm subdomains (5)(6)(7)(8)(9). Although the structures of the fingers and thumb subdomains vary considerably, the catalytic palm subdomains are all superimposable (10,11).…”

Section: Escherichia Coli Dna Polymerase I (Pol I)mentioning

confidence: 99%

The Conserved Active Site Motif A of Escherichia coliDNA Polymerase I Is Highly Mutable

Shinkai

Patel

Loeb

2001

Journal of Biological Chemistry

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Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val 700 -Arg 712 by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp 705 can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wildtype motif A sequence. As an example, we established that the naturally occurring Ile 709 has a previously undocumented role in supporting sugar discrimination.Escherichia coli DNA polymerase I (pol I) 1 is a multifunctional enzyme with roles in DNA replication, DNA repair, and genetic recombination (1). The first recognized and most thoroughly investigated of all DNA polymerases, it is key to our understanding of how DNA polymerases function as protein catalysts and as central enzymes in DNA metabolism. It belongs to one of six families of DNA polymerases, defined on the basis of amino acid sequence comparisons (2-4): family A (e.g. E. coli pol I, Thermus aquaticus (Taq) pol I, Bacillus stearothermophilus pol I, and T7 DNA polymerase), family B (e.g. DNA polymerase ␣ and RB69 DNA polymerase), reverse transcriptase (e.g. human immunodeficiency virus reverse transcriptase, and murine leukemia virus reverse transcriptase), family X (e.g. DNA polymerase ␤), the pol III family, and the UmuC/DinB family (e.g. DNA polymerase ). Crystal structures of representative enzymes from the first four families have been determined, revealing a common overall architecture that has been likened to a human right hand, with fingers, thumb, and palm subdomains (5-9). Although the structures of the fingers and thumb subdomains vary considerably, the catalytic palm subdomains are all superimposable (10, 11). The palm subdomain includes two conserved sequences, motif A and motif C, each harboring a catalytically essential aspartic acid residue. Essential roles of motif A in catalysis include interaction with the incoming dNTP and coordination with two divalent metal ions that are required for the polymerization reaction (12-15). Motif A begins at an anti-parallel ␤-strand containing predominantly hydrophobic residues and is followed by a turn and an ␣-helix. Although there is considerable variation in the amino acid sequence of the anti-parallel ␤-strand, the sequence of the turn and helix, DYSQIELR, is nearly invariant among known prokaryotic family A polymerases (16). 2In a recent study of Ta...

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Crystal Structure of Rat DNA Polymerase β: Evidence for a Common Polymerase Mechanism

Cited by 474 publications

References 48 publications

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

The Conserved Active Site Motif A of Escherichia coliDNA Polymerase I Is Highly Mutable

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