Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method.
1,N6-Ethenoadenine (epsilonA) is generated endogenously by lipid peroxidation and exogenously by tumorigenic industrial agents, vinyl chloride, and vinyl carbamate. epsilonA detected in human tissues causes mutation and is implicated in liver, colon and lung cancers. N-methyl purine DNA-glycosylase (MPG) is the only enzyme known so far to repair epsilonA. However, the mechanism of in vivo repair of epsilonA and the role of MPG remain enigmatic. Moreover, previous in vivo repair studies for DNA lesions, including epsilonA, focused only on the step of the removal of the base lesion without further insight into the completion of the repair process. This may be in part due to the unavailability of an appropriate in vivo quantitative method to evaluate complete BER process at the basal level. Our newly developed in vivo method is highly sensitive and involves phagemid M13mp18, containing epsilonA at a defined position. The complete repair events have been estimated by plaque assay in E. coli with the phagemids recovered from the human cells after cellular processing. We found that the detectable complete (removal and replacement of epsilonA with adenine) repair was observed only 18% in 16 h, but with the repair nearing completion within 24 h in colon cancer, HCT-116, cells. Moreover, MPG is the predominant enzyme for the BER process to remove epsilonA in mammalian cells. Although, the epsilonA is fairly a bulky adduct compared to other small BER substrate lesions, NER pathway is not involved in repair of this adduct. Furthermore, the epsilonA repair in vivo and in vitro is predominant in the G0/G1 phase of the cell cycle.
Choudhury
Sujata
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Adhikari
Sanjay
S
Cheema
Amrita
A
Roy
Rabindra
R
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002453:Cell Cycle; D002457:Cell Extracts; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D002457:Cell Extracts; D009153:Mutagens; C004981:1,N(6)-ethenoadenine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D000225:Adenine