Mechanism of Release and Fate of Excised Oligonucleotides during Nucleotide Excision Repair

A wide range of environmental and carcinogenic agents form bulky lesions on DNA that are removed from the human genome in the form of short, ∼30-nucleotide oligonucleotides by the process of nucleotide excision repair. Although significant insights have been made regarding the mechanisms of damage recognition, dual incisions, and repair resynthesis during nucleotide excision repair, the fate of the dual incision/excision product is unknown. Using excision assays with both mammalian cell-free extract and purified proteins, we unexpectedly discovered that lesion-containing oligonucleotides are released from duplex DNA in complex with the general transcription and repair factor, Transcription Factor IIH (TFIIH). Release of excision products from TFIIH requires ATP but not ATP hydrolysis, and release occurs slowly, with a t ½ of 3.3 h. Excised oligonucleotides released from TFIIH then become bound by the single-stranded binding protein Replication Protein A or are targeted by cellular nucleases. These results provide a mechanism for release and an understanding of the initial fate of excised oligonucleotides during nucleotide excision repair. Background: The fate of oligonucleotides released during nucleotide excision repair is unknown. Results: Damage-containing oligonucleotides are released from duplex DNA in complex with TFIIH but then slowly dissociate and bind RPA or are degraded by nucleases. Conclusion: Excised oligonucleotides form complexes with TFIIH and RPA during excision repair. Significance: Oligonucleotides released during nucleotide excision repair may function in the cellular response to DNA damage.