title: Deoxyinosine repair in nuclear extracts of human cells.
abstract: BACKGROUND
  Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mammalian cells appears more complex and was not fully understood.
  
  
  RESULTS
  All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofactors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair.
  
  
  CONCLUSIONS
  A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention.
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC.
authors.forename: Chia-Chia
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC ; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC.
authors.forename: Ya-Chien
authors.initials: YC
authors.lastname: Yang
authors.affiliation: Center for Microbial Pathogenesis, Nationwide Children's Hospital, Columbus, OH USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Goodman
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC.
authors.forename: Shi
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC.
authors.forename: Teng-Yung
authors.initials: TY
authors.lastname: Huang
authors.affiliation: Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC.
authors.forename: Wern-Cherng
authors.initials: WC
authors.lastname: Cheng
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC ; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC.
authors.forename: Liang-In
authors.initials: LI
authors.lastname: Lin
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC ; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC.
authors.forename: Woei-Horng
authors.initials: WH
authors.lastname: Fang
keywords: DNA repair deficiency; Deoxyinosine repair; Human cell extracts; In vitro assay; Mismatch repair