Vasquez Karen M, Christensen Jesper, Li Lei, Finch Rick A, Glazer Peter M
Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Park Road 1-C, Smithville, TX 78957, USA.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5848-53. doi: 10.1073/pnas.082193799. Epub 2002 Apr 23.
Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.
核苷酸切除修复（NER）通过检测和修复多种DNA损伤在维持基因组完整性方面发挥核心作用。着色性干皮病A互补组蛋白（XPA）是修复机制的重要组成部分，被认为作为DNA损伤识别和/或确认因子参与初始步骤。据报道，人类复制蛋白A（RPA）与XPA相互作用形成一种DNA损伤识别复合物，对受损DNA的特异性高于单独的XPA。这两种蛋白质识别由不同类型DNA损伤产生的如此广泛结构的机制尚不清楚。一种可能性是它们识别损伤的共同特征，例如螺旋主链的扭曲。我们通过确定人类XPA和RPA蛋白是否能识别由DNA三链螺旋诱导的螺旋扭曲来检验这一想法，DNA三链螺旋是一种非经典DNA结构，已被证明可诱导DNA修复、诱变和重组。我们测量了XPA和RPA一起或分别与含有通过补骨脂素共轭和光加成共价连接的三条、两条或无链三链体的底物的结合。我们发现单独的RPA能识别所有共价三链体结构，但在较高浓度下也会形成多价非特异性DNA聚集体。XPA自身不能识别底物，但在RPA存在时能结合它们。添加XPA可减少非特异性DNA聚集体的形成。这些结果支持了NER机制靶向螺旋扭曲的假设，并表明即使没有XPA时RPA也能识别受损DNA。
