title: Characterization of reaction intermediates of human excision repair nuclease.
abstract: Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: D
authors.initials: D
authors.lastname: Mu
authors.forename: M
authors.initials: M
authors.lastname: Wakasugi
authors.forename: D S
authors.initials: DS
authors.lastname: Hsu
authors.forename: A
authors.initials: A
authors.lastname: Sancar
mesh_terms: D001483:Base Sequence; D004260:DNA Repair; D004274:DNA, Recombinant / Q000737:chemistry; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D025842:SUMO-1 Protein; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D025842:SUMO-1 Protein; D014452:Ubiquitins; D004720:Endonucleases