Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage

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• 作者：Yu Liu ; Yiyong Liu ; Zhengguan Yang ; Christopher Utzat ; Guizhi Wang ; Ashis K. Basu ; and Yue Zou
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 17, 2005
• 年：2005
• 卷：44
• 期：19
• 页码：7361 - 7368
• 全文大小：358K
• 年卷期：v.44,no.19(May 17, 2005)
• ISSN：1520-4995

文摘

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excisionrepair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However,what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occursin terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purifiedfrom baculovirus-infected sf21 insect cells and the methods of gel mobility shift assays, gel filtrationchromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA boundto the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamagedDNA. The binding occurred in a sequential and protein concentration-dependent manner. At relativelylow-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higherconcentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopicand competitive binding analyses indicated that the specific binding of XPA to the adduct was significantlyfacilitated and stabilized by the presence of the second XPA in a positive cooperative manner. Thiscooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K₁ = 1.4 × 10⁶M^-1 and K₂ = 1.8 × 10⁷ M^-1. When interaction of XPA and RPA with DNA was studied, even thoughbinding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect onthe overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind toDNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of differenttypes of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.