Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage

Yu Liu; Yiyong Liu; Zhengguan Yang; Christopher Utzat; Guizhi Wang; Ashis K Basu; Yue Zou

doi:10.1021/bi047598y

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Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage

Journal article

Open access

Peer reviewed

Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage

Yu Liu, Yiyong Liu, Zhengguan Yang, Christopher Utzat, Guizhi Wang, Ashis K Basu and Yue Zou

Biochemistry (Easton), Vol.44(19), pp.7361-7368

05/17/2005

DOI: https://doi.org/10.1021/bi047598y

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https://hdl.handle.net/2376/116103

PMCID: PMC1475732

PMID: 15882075

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https://doi.org/10.1021/bi047598yView

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Abstract

Protein Binding - genetics

Humans

DNA Repair - radiation effects

DNA Adducts - metabolism

DNA-Binding Proteins - metabolism

Ultraviolet Rays

Protein Binding - radiation effects

DNA Adducts - radiation effects

Scintillation Counting

Replication Protein A

Dimerization

2-Acetylaminofluorene - radiation effects

Cross-Linking Reagents

DNA-Binding Proteins - radiation effects

DNA Replication

DNA-Binding Proteins - isolation & purification

DNA-Binding Proteins - chemistry

2-Acetylaminofluorene - metabolism

2-Acetylaminofluorene - analogs & derivatives

Binding, Competitive - genetics

DNA Adducts - genetics

Chromatography, Gel

Fluorescence Polarization

DNA Damage - radiation effects

Xeroderma Pigmentosum Group A Protein

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (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 occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.

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Title: Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage
Creators: Yu Liu - Department of Biochemistry and Molecular Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA
Yiyong Liu
Zhengguan Yang
Christopher Utzat
Guizhi Wang
Ashis K Basu
Yue Zou
Publication Details: Biochemistry (Easton), Vol.44(19), pp.7361-7368
Academic Unit: Biomedical Sciences, Department of
Publisher: United States
Grant note: R01 CA086927 / NCI NIH HHS ES00318 / NIEHS NIH HHS R56 ES009127 / NIEHS NIH HHS R56 CA086927 / NCI NIH HHS CA86927 / NCI NIH HHS K02 ES000318 / NIEHS NIH HHS R01 ES009127 / NIEHS NIH HHS ES09127 / NIEHS NIH HHS
Identifiers: 99900547657501842
Language: English
Resource Type: Journal article