Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA.
The xeroderma pigmentosum group A complementing protein (XPAC) is involved in an early step of nucleotide excision repair, the main process that removes UV damage and many chemical lesions from DNA. To explore the properties and function of XPAC, recombinant protein encoded by the human XPAC cDNA was expressed with an N-terminal polyhistidine tag in Escherichia coli and purified to homogeneity. The soluble fusion protein could correct the repair defect in vitro of XP-A cell extracts. XPAC protein bound to DNA with a preference for UV-irradiated over nonirradiated DNA, as determined by a gel electrophoresis mobility shift assay with a 258 base pair DNA fragment (the association constant was approximately 3 x 10(6) M-1 for the fragment irradiated with 6 kJ/m2 UV light). Removal of cyclobutane pyrimidine dimers from UV-irradiated DNA by enzymatic photoreactivation did not significantly reduce binding of XPAC to the irradiated fragment, indicating that binding was mostly due to (6-4) photoproducts, with a preference for a (6-4) photoproduct over an undamaged base pair up to 300-fold. Undamaged single-stranded DNA competed about 4-fold more effectively than undamaged double-stranded DNA for binding of XPAC to a UV-irradiated fragment. In addition, XPAC bound to DNA treated with the chemotherapeutic agent cis-diamminedichloroplatinum(II). The results suggest that XPAC functions as a key component in recognition of DNA damage during repair.
Jones
C J
CJ
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, U.K.
Wood
R D
RD
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism*; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA