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

ABSTRACT: DNA polymerase ? (Pol ?) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol ? incorporates a nucleotide into a single-gapped DNA substrate. Pol ? 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 ? 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 ? undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol ? 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 ? 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 ?. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol ?.

SUBMITTER: Huang J 

PROVIDER: S-EPMC6166726 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature