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

• Published in: The Journal of biological chemistry 2018-09, Vol.293 (39), p.15084-15094
• Main Authors: Huang, Ji, Alnajjar, Khadijeh S., Mahmoud, Mariam M., Eckenroth, Brian, Doublié, Sylvie, Sweasy, Joann B.
• Format: Article
• Language: English
• Subjects: 5′-phosphate; Catalysis; conformational change; crystal structure; Crystallography, X-Ray; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA End-Joining Repair - genetics; DNA polymerase; DNA Polymerase beta - chemistry; DNA Polymerase beta - genetics; DNA Repair - genetics; DNA Replication - genetics; Enzymology; FRET; gap size; Humans; Kinetics; Nucleotides - chemistry; Nucleotides - genetics; polymerase β; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.004564

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.