Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.
Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.
Kuraoka
I
I
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Robins
P
P
Masutani
C
C
Hanaoka
F
F
Gasparutto
D
D
Cadet
J
J
Wood
R D
RD
Lindahl
T
T
D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D005609:Free Radicals; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D005609:Free Radicals; D009841:Oligonucleotides; D017382:Reactive Oxygen Species; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases