Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.
Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.
复制标题
生成位点特异性标记的荧光人 XPA，用于研究核苷酸切除修复过程中的 DNA 结合动力学。
DOI：
10.1016/j.ymeth.2024.02.006
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Antony,Edwin
中科院分区：
文献类型：
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作者：
Kuppa,Sahiti;Corless,Elliot;Caldwell,ColleenC;Spies,Maria;Antony,Edwin
核苷酸切除修复（NER）通过去除由外部因素如紫外线引入的大体积DNA加合物来促进基因组完整性。NER酶的缺陷与病理状况如着色性干皮病、硫营养不良和Cockayne综合征相关。NER中的关键步骤是着色性干皮病A组蛋白（XPA）与ss/ds DNA连接点的结合。为了更好地捕获NER期间XPA与DNA相互作用的动力学，我们利用了通过非典型氨基酸（FEncAA）方法的荧光增强。将4-叠氮基-L-苯丙氨酸（4AZP或pAzF）掺入人XPA中的Arg-158处，并使用应变促进的叠氮化物-炔环加成与Cy 3缀合。所得荧光XPA蛋白（XPAC γ 3）显示DNA结合活性无损失，并在结合DNA时产生强烈的荧光变化。在这里，我们描述的方法，以产生XPACy 3和详细的体外实验条件下所需的稳定保持蛋白质在生化和生物物理研究。
Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3and detail invitroexperimental conditions required to stably maintain the protein during biochemical and biophysical studies.