Premium
Nuclear Extracts Enzymatically Convert M 1 dG to 6‐oxo‐M 1 dG in Duplex DNA
Author(s) -
Mitchener Michelle M.,
Wauchope Orrette R.,
Marnett Lawrence J.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.573.3
Subject(s) - chemistry , enzyme , oligonucleotide , dna , ferrous , biochemistry , nucleic acid , microbiology and biotechnology , biology , organic chemistry
Lipid and DNA peroxidation give rise to DNA adducts, the most abundant of which is 3‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)pyrimido[1,2‐α]purin‐10(3H)‐one (M 1 dG). Increased levels of this adduct are positively correlated with a variety of physiological and pathophysiological conditions including aging, bacterial infection, and cancer. Recently, our laboratory reported the oxidative metabolism of M 1 dG to 6‐oxo‐M 1 dG in nuclear DNA isolated from multiple human and rodent cell lines, suggesting the presence of an activity responsible for conversion of M 1 dG to 6‐oxo‐M 1 dG in double‐stranded DNA. Nuclear extracts from RAW264.7 cells were prepared and incubated with double‐stranded oligonucleotides containing M 1 dG. Following incubation, the oligonucleotides were digested and analyzed for the presence of 6‐oxo‐M 1 dG. Heat‐denaturation or Proteinase K‐treatment of the nuclear lysates ablated conversion, indicating that the oxidation is an enzyme‐mediated process. We hypothesized that this enzyme might be an alpha‐ketoglutarate‐dependent dioxygenase, a class of iron‐dependent enzymes that is responsible for the oxidation of various alkylated nucleic acids and/or amino acids. Indeed, we found that preincubation of nuclear lysates with EDTA or metals known to displace ferrous ion from proteins, resulted in a decrease in 6‐oxo‐M 1 dG formation upon incubation of the lysates with the M 1 dG‐containing oligonucleotide. Conversely, preincubation with ferrous ion, but not ferric ion, increased the amount of 6‐oxo‐M 1 dG formed. Additionally, nuclear lysates preincubated with either N ‐oxalylglycine or IOX1, two structurally distinct broad‐spectrum alpha‐ketoglutarate‐dependent dioxygenase inhibitors, showed reduced 6‐oxo‐M 1 dG formation compared to their control counterparts. These findings indicate the presence of a nuclear enzyme, conceivably an alpha‐ketoglutarate‐dependent dioxygenase, capable of converting M 1 dG to 6‐oxo‐M 1 dG in double‐stranded DNA. Support or Funding Information This work was supported by grants from the National Institutes of Health (R37 CA0878819 to L.J.M.) and US Public Health Services (T32 ES007028 to M.M.M.).