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. 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.
Highlights
Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine
Deoxyinosine in DNA can arise from spontaneous deamination of deoxyadenosine residue, and is induced by ROS produced from normal aerobic respiration
Deoxyinosine‐containing substrates are efficiently processed in Hela cell extracts Previously, to evaluate Endonuclease V (EndoV) repair system in E. coli, we constructed a set of dI-containing substrates A-I, G-I and T-I heteroduplexes [9, 11]
Summary
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 radia‐ tion, UV light, nitrous acid, or heat. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. Deoxyinosine (hypoxanthine deoxyribonucleotide, dI) in DNA can arise from spontaneous deamination of deoxyadenosine residue, and is induced by ROS produced from normal aerobic respiration. Deoxyinosine derived from deamination of deoxyadenosine in DNA is potentially mutagenic since it prefers to pair with dCTP. Base excision repair (BER) was thought to be the major pathway for dI repair. From radionucleotide incorporation fine mapping, the resulting apurinic/apyrimidinic (AP) sites are further processed by both the short patch pathway (1-nucleotide gap filling) with DNA polymerase (Pol) β and the long patch pathway (2-6 nucleotide resynthesis) with Pol δ and PCNA [7]
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