Jaiswal Aruna S, Williamson Elizabeth A, Jaiswal Arunima S, Kong Kimi, Hromas Robert A
Division of Hematology and Medical Oncology, Department of Medicine and the Mays Cancer Center, University of Texas Health Science Center, San Antonio, TX, USA.
Division of Hematology and Oncology, Department of Medicine, University of Texas Health Science Center, San Antonio, TX, USA.
Methods Mol Biol. 2023;2701:91-112. doi: 10.1007/978-1-0716-3373-1_6.
The mammalian cell genome is continuously exposed to endogenous and exogenous insults that modify its DNA. These modifications can be single-base lesions, bulky DNA adducts, base dimers, base alkylation, cytosine deamination, nitrosation, or other types of base alteration which interfere with DNA replication. Mammalian cells have evolved with a robust defense mechanism to repair these base modifications (damages) to preserve genomic stability. Base excision repair (BER) is the major defense mechanism for cells to remove these oxidative or alkylated single-base modifications. The base excision repair process involves replacement of a single-nucleotide residue by two sub-pathways, the single-nucleotide (SN) and the multi-nucleotide or long-patch (LP) base excision repair pathways. These reactions have been reproduced in vitro using cell free extracts or purified recombinant proteins involved in the base excision repair pathway. In the present chapter, we describe the detailed methodology to reconstitute base excision repair assay systems. These reconstitutive BER assay systems use artificially synthesized and modified DNA. These reconstitutive assay system will be a true representation of biologically occurring damages and their repair.
哺乳动物细胞基因组不断受到内源性和外源性损伤，这些损伤会对其DNA进行修饰。这些修饰可以是单碱基损伤、大分子DNA加合物、碱基二聚体、碱基烷基化、胞嘧啶脱氨基、亚硝化或其他干扰DNA复制的碱基改变类型。哺乳动物细胞进化出了强大的防御机制来修复这些碱基修饰（损伤），以维持基因组稳定性。碱基切除修复（BER）是细胞去除这些氧化或烷基化单碱基修饰的主要防御机制。碱基切除修复过程通过两个子途径，即单核苷酸（SN）和多核苷酸或长补丁（LP）碱基切除修复途径，来替换单个核苷酸残基。这些反应已在体外使用无细胞提取物或参与碱基切除修复途径的纯化重组蛋白进行了重现。在本章中，我们描述了重建碱基切除修复检测系统的详细方法。这些重建的BER检测系统使用人工合成和修饰的DNA。这些重建检测系统将真实反映生物学上发生的损伤及其修复情况。
