The Hydrophobic Hinge Region of Rat DNA Polymerase β Is Critical for Substrate Binding Pocket Geometry

Daniela Starcevic,Shibani Dalal,Joachim Jaeger,Joann B Sweasy

doi:10.1074/jbc.m502178200

Daniela Starcevic, Shibani Dalal + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m502178200

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Abstract

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.

Highlights

Summary and Implications—Our data show that changing hinge residue 260 of Pol␤ from Ile to Gln results in a polymerase that has nearly wild type activity but is a mutator mutant
We find that the kinetic basis for misincorporation by I260Q lies in loss of discrimination at the level of dNTP substrate binding
Our modeling studies indicate that the Gln mutant affects the domain closure around the DNA substrate, that the protein matrix around the ribose moiety of the incoming nucleotide is altered, and that the DNA template could be positioned differently in this variant than in the wild type enzyme (19)

Summary

The abbreviation used is

Given the known role of polymerase ␤ in DNA repair and the possible role in meiosis as well as its implicated roles in small-scale replicative synthesis, it is of crucial importance to understand the types and nature of mutations that could lead to infidelity. This infidelity can result in the accumulation of mutations that could, in turn, result in cancer and/or abnormalities in cellular processes. Our results suggest that the hinge of Pol␤ is important for discrimination during the binding of nucleotide substrate

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