The E295K cancer variant of human polymerase β favors the mismatch conformational pathway during nucleotide selection

Brian E. Eckenroth, Jamie B. Towle-Weicksel, Joann B. Sweasy, Sylvie Doublié

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damagedDNAas part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (~30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for bindingdCTPopposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.

Original languageEnglish (US)
Pages (from-to)34850-34860
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number48
DOIs
StatePublished - Nov 29 2013
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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