基底切除修复术
分子生物学
DNA糖基化酶
质粒
DNA
AP站点
转染
限制地点
生物
碱基对
AP核酸内切酶
转化(遗传学)
化学
限制性酶
DNA损伤
基因
遗传学
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 3-19
标识
DOI:10.1007/978-1-0716-3373-1_1
摘要
The base excision repair (BER) pathway repairs small, non-bulky DNA lesions, including oxidized, alkylated, and deaminated bases, and is responsible for the removal of at least 20,000 DNA lesions per cell per day. BER is initiated by DNA damage-specific DNA glycosylases that excise the damaged base and generates an abasic (AP) site or single-strand breaks, which are subsequently repaired in mammalian cells either by single-nucleotide (SN) or multiple-nucleotide incorporation via the SN-BER or long-patch BER (LP-BER) pathway, respectively. This chapter describes a plaque-based host cell reactivation (PL-HCR) assay system for measuring BER mechanisms in live mammalian cells using a plasmid-based assay. After transfection of a phagemid (M13mp18) containing a single modified base (representative BER DNA substrates) within a restriction site into human cells, restriction digestions detect the presence or absence (complete repair) of the adduct by the transformation of the digestion products into E. coli and counting the transformants as plaques. To monitor the patch size, different plasmids are constructed containing C:A mismatches within different restriction sites (inhibiting digestion) at various distances on both sides (5' or 3') of the modified base-containing restriction sites. Using this assay, the percentage of repair events that occur via 5' and 3' patch formation can be quantified.
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