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

Ji Huang, Khadijeh S. Alnajjar, Mariam M. Mahmoud, Brian Eckenroth, Sylvie Doublie, Joann B. Sweasy

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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 Polβ 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.

Original languageEnglish (US)
Pages (from-to)15084-15094
Number of pages11
JournalJournal of Biological Chemistry
Volume293
Issue number39
DOIs
StatePublished - Sep 28 2018
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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