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Biochemistry. 2017 Oct 10;56(40):5449-5456. doi: 10.1021/acs.biochem.7b00713. Epub 2017 Sep 20.

DNA Polymerase β Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the β-γ Bridging Group of the Incoming dNTP.

Author information

1
Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
2
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.

Abstract

The hydrophobic hinge region of DNA polymerase β (pol β) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol β variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol β, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the β-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.

PMID:
28862868
PMCID:
PMC5634933
DOI:
10.1021/acs.biochem.7b00713
[Indexed for MEDLINE]
Free PMC Article

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