CHARACTERIZATION OF REACTION INTERMEDIATES OF HUMAN EXCISION-REPAIR NUCLEASE

Nucleotide excision repair in humans is a complex reaction involving 1 4 polypeptides in six repair factors for dual incisions on either side s of a DNA lesion, To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: puri fication of functional DNA protein complexes, permanganate footprintin g, and the employment as substrate of presumptive DNA reaction interme diates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by s ubstrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision compl ex 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 func tion, the XPG protein stabilized this complex, forming a long lived pr eincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, formi ng PIC3, which led to dual incisions and the release of the excised ol igomer, With partially unwound DNAs, thymine cyclobutane dimer was exc ised 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 le sion unwinding in preincision complexes.