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J Biol Chem. 2018 Sep 28;293(39):15084-15094. doi: 10.1074/jbc.RA118.004564. Epub 2018 Aug 1.

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

Author information

1
From the Departments of Therapeutic Radiology and.
2
the Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
3
From the Departments of Therapeutic Radiology and joann.sweasy@yale.edu.
4
Genetics, Yale University School of Medicine, New Haven, Connecticut 06520 and.

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 Pol β 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.

KEYWORDS:

5′-phosphate; DNA polymerase; FRET; conformational change; crystal structure; gap size; kinetics; polymerase β

PMID:
30068550
PMCID:
PMC6166726
[Available on 2019-09-28]
DOI:
10.1074/jbc.RA118.004564

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