Deoxyinosine repair in nuclear extracts of human cells
Abstract
BackgroundDeamination 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.ResultsAll 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.ConclusionsA 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.…
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