Oxidation of 7,8-dihydro-8-oxoguanine affords lesions that are potent sources of replication errors in vivo.
Three single-stranded DNA genomes have been constructed that contain the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) oxidation products oxaluric acid, oxazalone, and cyanuric acid. Oligonucleotides containing each lesion were synthesized by treating an oligonucleotide containing a single 8-oxodG with peroxynitrite, and the desired products were isolated by HPLC. The modified oligonucleotides were ligated into M13mp7L2 bacteriophage DNA in such a way that the lesion was situated at a known site in the lacZ gene fragment of the viral genome. The circular genomes were transfected into wild-type AB1157 Escherichia coli. The relative efficiency of lesion bypass by DNA polymerase was determined by counting the number of initial independent infections produced by each genome relative to that of an unmodified DNA control. Viral progeny were analyzed for mutation frequency and type by PCR amplification of the insert region followed by a recently developed post-labeling assay. All three secondary lesions were readily bypassed, causing G --> T transversions at frequencies at least an order of magnitude higher than 8-oxodG. These data establish a model whereby the modestly mutagenic primary lesion 8-oxodG is oxidized in vivo to much more highly mutagenic secondary lesions.
Henderson
Paul T
PT
Division of Bioengineering and Environmental Health and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Delaney
James C
JC
Gu
Feng
F
Tannenbaum
Steven R
SR
Essigmann
John M
JM
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D004249:DNA Damage; D017931:DNA Primers; D004261:DNA Replication; D004270:DNA, Circular / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine