Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1

Chem Res Toxicol. 2016 Mar 21;29(3):367-79. doi: 10.1021/acs.chemrestox.5b00513. Epub 2016 Mar 4.

Abstract

The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g., apurinic/apyrimidinic (AP) sites, N(2)-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330-833) proteins and DNA templates containing a G, AP site, N(2)-CH2(2-naphthyl)G (N(2)-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in kcat/Km for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N(2)-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than the wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.

Publication types

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

MeSH terms

  • DNA / chemistry*
  • DNA / metabolism*
  • DNA Adducts / chemistry
  • DNA Damage*
  • G-Quadruplexes*
  • Germ-Line Mutation / genetics*
  • Humans
  • Models, Molecular
  • Mutation, Missense / genetics*
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / genetics*
  • Nucleotidyltransferases / metabolism*

Substances

  • DNA Adducts
  • Nuclear Proteins
  • DNA
  • Nucleotidyltransferases
  • REV1 protein, human