Substrate-induced DNA Polymerase β Activation

Beard, William A.; Shock, David D.; Batra, V.K.; Prasad, Rajendra; Wilson, Samuel H.

doi:10.1074/jbc.m114.607432

Cited by 25 publications

(28 citation statements)

References 59 publications

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“…To measure the rate of the first insertion ( k pol ) and apparent equilibrium nucleotide dissociation constant ( K d ), single-turnover kinetic assays (enzyme/DNA = 10) were performed as described previously 46 . Briefly, a pre-incubated solution of enzyme/DNA was rapidly mixed with various concentrations of MgCl 2 /dGMPPNP using a Kintek RQF-3 rapid quench-flow.…”

Section: Methodsmentioning

confidence: 99%

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

et al. 2017

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DNA polymerases catalyze efficient and high fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, removing the DNA primer terminus and generating deoxynucleoside triphosphates. Since pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that it was limited by a non-chemical step. Utilizing a pyrophosphate analog, where the bridging oxygen is replaced with an imido-group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium that favored the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.

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Section: Methodsmentioning

confidence: 99%

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

et al. 2017

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“…Structural and biochemical data support the hypothesis that some DNA polymerases discriminate between alternate dNTP substrates through an “induced fit” mechanism where binding of the correct nucleotide leads to substrate/protein conformational adjustments that align catalytic groups to optimize chemistry [39–43]. Recently, time-lapse X-ray crystallography studies using natural substrates revealed high-resolution structures of novel catalytic intermediates within the pol β active site [44–46].…”

Section: Impact Of Pol β Structural Conformations On Channeling Dnmentioning

confidence: 99%

Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

Çağlayan¹,

Wilson²

2015

DNA Repair

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DNA lesions arise from many endogenous and environmental agents, and they promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5′-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.

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“…The catalytic and nucleotide magnesium ions are shown as orange spheres. Panels B and C were adapted from Beard et al [29]. …”

Section: Figmentioning

confidence: 99%

New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

2015

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Time-lapse X-ray crystallography allows visualization of intermediate structures during the DNA polymerase catalytic cycle. Employing time-lapse crystallography with human DNA polymerase β has recently allowed us to capture and solve novel intermediate structures that are not stable enough to be analyzed by traditional crystallography. The structures of these intermediates reveal exciting surprises about active site metal ions and enzyme conformational changes as the reaction proceeds from the ground state to product release. In this perspective, we provide an overview of recent advances in understanding the DNA polymerase nucleotidyl transferase reaction and highlight both the significance and mysteries of enzyme efficiency and specificity that remain to be solved.

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Substrate-induced DNA Polymerase β Activation

Cited by 25 publications

References 59 publications

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

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