Lao Y, Gomes X V, Ren Y, Taylor J S, Wold M S
Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
Biochemistry. 2000 Feb 8;39(5):850-9. doi: 10.1021/bi991704s.
Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.
人类复制蛋白A（RPA）是一种异源三聚体单链DNA结合蛋白（亚基分子量分别为70、32和14 kDa），是细胞DNA代谢所必需的。据报道，RPA可与受损双链DNA特异性相互作用，并参与核苷酸切除修复（NER）的多个步骤，包括损伤识别步骤。我们研究了RPA与含有损伤的单链和双链DNA（分别为ssDNA和dsDNA）结合的机制。我们发现，RPA对受损dsDNA的亲和力与受损碱基对双螺旋的破坏相关，并且需要RPA的ssDNA结合活性。我们得出结论，RPA识别的是由受损核苷酸导致的单链特征。我们还表明，RPA可特异性结合受损的ssDNA。结合的特异性因损伤类型而异，RPA对嘧啶（6-4）嘧啶酮光产物的偏好性高达60倍。我们发现这种特异性结合绝对依赖于70 kDa亚基C端区域的锌指结构域。RPA对受损ssDNA的亲和力比损伤识别蛋白XPA（着色性干皮病A组蛋白）高5个数量级。这些发现表明，RPA可能在NER切除复合物中与受损和未受损的链都结合。RPA的结合可能对NER中受损DNA的有效切除很重要。
