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 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.
Cell Biosci. 2015 Sep 8;5:52. doi: 10.1186/s13578-015-0044-8. eCollection 2015.
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.
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.
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.
在生理条件下，腺嘌呤的脱氨作用会自发发生，生成高度诱变的损伤——次黄嘌呤。这一过程会因 DNA 暴露于电离辐射、紫外线、亚硝酸盐或热而产生的 ROS 而增强。DNA 中的次黄嘌呤可以与胞嘧啶配对，导致 DNA 复制后 A:T 到 G:C 的转换突变。在大肠杆菌中，脱氧肌苷（次黄嘌呤脱氧核苷酸，dI）通过内切酶 V 启动的替代切除修复途径被去除。然而，哺乳动物细胞中 dI 的纠正似乎更为复杂，尚未完全理解。
将包括 A-I、C-I、G-I 和 T-I 在内的所有四种可能的 dI 含有异源双链 DNA 引入含有人细胞提取物的修复反应中。修复反应需要镁、dNTP 和 ATP 作为辅助因子。我们发现 G-I 是最好的底物，其次是 T-I、A-I 和 C-I。此外，从修复要求和对特定聚合酶抑制剂的敏感性来看，在 DNA 中处理 dI 时存在重叠的修复活性。事实上，一种遗传性非息肉病结直肠癌细胞系（HCT116）显示出较低的 dI 修复活性，部分归因于缺乏错配修复。
创建了一种基于质粒的方便且非放射性同位素方法来研究人细胞中的 dI 修复。可以通过限制性内切酶切割来评估体外处理的诱变 dI 损伤，以评分修复。本研究中描述的修复测定法为进一步研究人类修复途径在 dI 处理中的作用及其在突变预防中的生物学意义提供了良好的平台。
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