Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage.
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.
Liu
Yu
Y
Department of Biochemistry and Molecular Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Liu
Yiyong
Y
Yang
Zhengguan
Z
Utzat
Christopher
C
Wang
Guizhi
G
Basu
Ashis K
AK
Zou
Yue
Y
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000378:metabolism / Q000528:radiation effects; D001667:Binding, Competitive / Q000235:genetics; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D019281:Dimerization; D005454:Fluorescence Polarization; D006801:Humans; D011485:Protein Binding / Q000235:genetics / Q000528:radiation effects; D051716:Replication Protein A; D012588:Scintillation Counting; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D015073:2-Acetylaminofluorene