REACTION-MECHANISM OF HUMAN DNA-REPAIR EXCISION NUCLEASE

Nucleotide excision repair consists of removal of the damaged nucleoti de(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. W e have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/su bstrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of t he thymine dimer substrate. However, when a cholesterol-substituted ol igonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely req uired for both incisions. The XPG subunit is essential to the formatio n of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF-ERCC1. Kine tic experiments revealed that the 3'-incision precedes the 5'-incision . Consistent with the kinetic data, uncoupled 5'-incision was never ob served in the reconstituted system. Two forms of TFIIH were used in th e reconstitution reaction, one containing the CDK7-cyclin H pair and o ne lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions oc curring during damage removal by human excision nuclease.