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

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


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Catalysis
  • Crystallography, X-Ray
  • DNA / biosynthesis*
  • DNA / chemistry
  • DNA / genetics
  • DNA End-Joining Repair / genetics
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / genetics
  • DNA Repair / genetics
  • DNA Replication / genetics*
  • Humans
  • Kinetics
  • Nucleotides / chemistry
  • Nucleotides / genetics*
  • Substrate Specificity


  • Nucleotides
  • DNA
  • DNA Polymerase beta

Associated data

  • PDB/2FMS
  • PDB/3ISB
  • PDB/2BPG