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

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.

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

“…This result is consistent with the existence of two different binding modes for WT and 3DKA on 1-nt-gapped DNA. Indeed, FRET studies of pol b binary complexes in the presence and absence of a 5´-phosphate have different FRET efficiencies, suggesting differential binding modes (22).…”

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

“…DNA polymerase ␤ (pol ␤) is an established mammalian model for studying nucleotide discrimination because of its amenability to structure-function studies and conformational nucleotide selection mechanism akin to replicative polymerases (12,13). The conformational change occurs upon correct nucleotide binding, where the polymerase N-subdomain closes to facilitate key nucleic acid-protein interactions for efficient catalysis (12)(13)(14)(15)(16)(17)(18). Following closure, catalysis is carried out by nucleophilic attack of the primer terminus oxyanion (O3Ј) on the ␣-phosphate (␣P) of the incoming nucleotide, resulting in deoxynucleoside monophosphate incorporation and generation of a pyrophosphate (19).…”

Human Polβ Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis

“…The analysis of the gel images allowed us to determine the dependence of the DNA-bound fraction on the enzyme concentration ( Figure 5 ), and this dependence was utilized to calculate the dissociation constants K d using Equation (1) ( Table 2 ). Unexpectedly, the dissociation constants for the wild-type protein were in the 0.33–0.59 μM range, that is, ~tenfold higher than expected for the K d values of Polβ (60 nM [ 51 ] 30 nM [ 48 ], 5 nM [ 33 ]). However, such a discrepancy of K d values most likely was achieved owing to the different length and type of the DNA substrates used in these studies as well as the difference in experimental conditions (pH and ionic strength of the buffer solution).…”