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A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity.

ABSTRACT: DNA polymerase beta (pol beta) is the main polymerase involved in the base excision repair pathway responsible for repairing damaged bases in the DNA. Previous studies on the H285D mutant of pol beta suggested that the C-terminal region of the polymerase is important for polymerase function. In this study, the C-terminal region of pol beta was mutated to assess its role in polymerization. Kinetic experiments showed that the C-terminal region is required for wild-type polymerase activity. Additionally, an interaction between the fingers and palm subdomain revealed itself to be required for polymerase activity. The E316R mutant of pol beta was shown to have a 29,000-fold reduction in polymerization rate with no reduction in nucleotide binding, suggesting that there exists a noncovalent mechanistic step between nucleotide binding and nucleophilic attack of the primer 3'-hydroxyl group on the ?-PO(4) of the nucleotide. Molecular modeling studies of the E316R mutant demonstrate that disrupting the interaction between Arg182 and Glu316 disrupts the packing of side chains in the hydrophobic hinge region and may be hampering the conformational change during polymerization. Taken together, these data demonstrate that the triad interaction of Arg182, Glu316, and Arg333 is crucial for polymerase function.

SUBMITTER: Murphy DL

PROVIDER: S-EPMC3601662 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity.

Murphy Drew L DL Jaeger Joachim J Sweasy Joann B JB

Journal of the American Chemical Society 20110331 16

DNA polymerase beta (pol beta) is the main polymerase involved in the base excision repair pathway responsible for repairing damaged bases in the DNA. Previous studies on the H285D mutant of pol beta suggested that the C-terminal region of the polymerase is important for polymerase function. In this study, the C-terminal region of pol beta was mutated to assess its role in polymerization. Kinetic experiments showed that the C-terminal region is required for wild-type polymerase activity. Additio ...[more]

PMID: 21452873

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Project description:BackgroundThe discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called 'Point-of-Care tests'. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited.ResultsWe have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8-9 and 25-110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25-37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes.ConclusionsSubstituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

Project description:XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.

Project description:Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the "open" configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

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A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity.

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A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity.

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