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

Shock, David D.; Freudenthal, Bret; Beard, William A.; Wilson, Samuel H.

doi:10.1038/nchembio.2450

Cited by 25 publications

(38 citation statements)

References 49 publications

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“…Recently, a study on DNA polymerase β has shown that utilization of dNTPs that are non-hydrolyzable at the β-γ position leads to the promotion of the pyrophosphorolysis reaction instead of the DNA synthesis reaction ( 49 ). Also, the utilization of a PPi analog, with an imido- linkage has been shown to promote pyrophosphorolysis ( 50 ).…”

Section: Discussionmentioning

confidence: 99%

Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction

Kottur

Nair

2018

Nucleic Acids Research

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DNA synthesis by DNA polymerases (dPols) is central to duplication and maintenance of the genome in all living organisms. dPols catalyze the formation of a phosphodiester bond between the incoming deoxynucleoside triphosphate and the terminal primer nucleotide with the release of a pyrophosphate (PPi) group. It is believed that formation of the phosphodiester bond is an endergonic reaction and PPi has to be hydrolyzed by accompanying pyrophosphatase enzymes to ensure that the free energy change of the DNA synthesis reaction is negative and it can proceed in the forward direction. The fact that DNA synthesis proceeds in vitro in the absence of pyrophosphatases represents a long-standing conundrum regarding the thermodynamics of the DNA synthesis reaction. Using time-resolved crystallography, we show that hydrolysis of PPi is an intrinsic and critical step of the DNA synthesis reaction catalyzed by dPols. The hydrolysis of PPi occurs after the formation of the phosphodiester bond and ensures that the DNA synthesis reaction is energetically favorable without the need for additional enzymes. Also, we observe that DNA synthesis is a two Mg2+ ion assisted stepwise associative SN2 reaction. Overall, this study provides deep temporal insight regarding the primary enzymatic reaction responsible for genome duplication.

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Section: Discussionmentioning

confidence: 99%

Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction

Kottur

Nair

2018

Nucleic Acids Research

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“…The poor performance of Pol ␤ on these DNA substrates might facilitate hand-off to other BER or alternative repair pathway components, but we currently do not have data to support such a conclusion. Studies from Joyce et al (20), Tsai and co-workers (21), Kellinger and Johnson (22), and our group (29) show that the pre-catalytic conformational step, including the reverse step, affects the fidelity of several polymerases. Therefore, the altered conformational change rates for DNA substrates other than the single-nucleotide gapped DNA might affect the fidelity of Pol ␤.…”

Section: Resultsmentioning

confidence: 61%

“…We proposed that the second NCS may be involved in the binding of catalytic metal into the Pol ␤ active site (the NCS throughout the text refers in particular to the second NCS step) (38). Using this system, we have also discovered that nucleotide release before chemistry plays an important role in the fidelity of Pol ␤, similar to what has been found for HIV reverse transcriptase (22,24,29). To characterize the nature of pre-catalytic conformational changes of Pol ␤ with a variety of DNA substrates, we performed FRET analysis of Pol ␤ on various DNA substrates that vary in both the gap size and the modification of 5Ј termini on the downstream DNA.…”

mentioning

confidence: 60%

“…by an increase in the fluorescence signal ( Fig. 5) (22,29). The trapping of dNTP will trigger the reverse conformational changes, as illustrated below,…”

Section: Dna Substrate Influences Conformational Changes Of Pol ␤mentioning

confidence: 92%

“…Specifically, the fingers domain of the polymerase closes onto the DNA substrate, aligning the terminal 3Ј-OH for the nucleophilic attack on the P␣ of the incoming nucleotide to form a phosphodiester bond, which is coordinated through a two-or perhaps three-metal ion-dependent mechanism (5,28). These conformational changes have been linked to polymerase fidelity (20,22,29). After the chemistry step, at least one additional conformational step is likely to facilitate the release of pyrophosphate (30, 31).…”

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confidence: 99%

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The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase β

Huang

Alnajjar

Mahmoud

et al. 2018

Journal of Biological Chemistry

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DNA polymerase β (Pol β) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol β incorporates a nucleotide into a single-gapped DNA substrate. Pol β may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5'-modified downstream DNA. We have recently shown that Pol β fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol β undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol β with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5'-phosphate group has a more significant effect. The absence of the 5'-phosphate decreases the DNA binding affinity of Pol β and results in a conformationally more open binary complex. Moreover, upon addition of the correct nucleotide in the absence of 5'-phosphate, a slow fingers closing step is observed. Interestingly, either increasing the gap size or removing the 5'-phosphate group results in loss of the noncovalent step. Together, these results suggest that the character of the DNA substrate impacts the nature and rates of pre-catalytic conformational changes of Pol β. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol β.

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Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance

Zhao,

Xu,

Yang

et al. 2024

Advanced Science

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DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3′‐OH deprotonation and the PPi formation are not totally clear. In this work, a method is presented to detect the full catalytic cycle of human Pol (hPol β) in graphene‐molecule‐graphene single‐molecule junctions. Real‐time in situ monitoring successfully revealed the spatial and temporal properties of the open and closed conformation states of hPol β, distinguishing the reaction states in the Pols catalytic cycle and unveiling 3′‐OH deprotonation and pyrophosphate (PPi) formation mechanism of hPol β. Proton inventory experiment demonstrated that the rate‐limiting step of PPi formation is deprotonation, which occurs before a reverse conformational change. Additionally, by detecting the acidity (pKa), it is found that MgA‐bound OH− acted as a general base and activated the nucleophile of 3′‐OH, and that acidic residue D190 or D192 coordinated with MgB as a proton donor to PPi. This work provides useful insights into a fundamental chemical reaction that impacts genome synthesis efficiency and Pol fidelity, which the discovery of Pol‐targeting drugs and design of artificial Pols for DNA synthetic applications are expected to accelerated.

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

Cited by 25 publications

References 49 publications

Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction

Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction

The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase β

Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single‐Molecule Conductance

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