Replication Protein A Interactions with DNA. III. Molecular Basis of Recognition of Damaged DNA

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• 作者：Ye Lao ; Xavier V. Gomes ; Yingjie Ren ; John-Stephen Taylor ; and Marc S. Wold
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：February 8, 2000
• 年：2000
• 卷：39
• 期：5
• 页码：850 - 859
• 全文大小：202K
• 年卷期：v.39,no.5(February 8, 2000)
• ISSN：1520-4995

文摘

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 reportedto interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotideexcision repair (NER) including the damage recognition step. We have examined the mechanism of RPAbinding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containingdamage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the doublehelix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA isrecognizing single-stranded character caused by the damaged nucleotides. We also show that RPA bindsspecifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPAhaving up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that thisspecific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDasubunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of thedamage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest thatRPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA bindingmay be important for efficient excision of damaged DNA in NER.