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

Abstract

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.

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

“…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 ).…”

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

“…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 ␤.…”

“…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.…”

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

“…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).…”