DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

Abstract

Background:The mechanism of excised oligonucleotide processing during nucleotide excision repair is unknown. Results: UV photoproduct-containing oligonucleotides associate with chromatin following the dual incisions. Inhibition of gap-filling activities results in an accumulation of RPA-bound small, excised, damaged DNA (sedDNA) fragments. Conclusion: Gap filling-mediated dissociation of sedDNAs from RPA influences nucleotide excision repair rate. Significance: sedDNA processing is important in the DNA damage response.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells

“…Simple Method for Monitoring DNA Repair, Checkpoint, and Apoptosis from a Single Cell Culture-We have previously described a method for isolation and quantification of excised oligonucleotides (nominal 30-mers) generated in vivo by nucleotide excision repair using a variety of cell lysis methods (7,8,13). We have expanded the utility of this assay by using a milder FIGURE 1.…”

“…The method consists of observing the nucleotide excision repair-specific ϳ30-nt-long oligomers ("nominal 30-mers," which are actually in the range of 24 -32 nucleotides) that had previously only been visualized in vitro (9 -12). The method involves cell lysis followed by immunoprecipitation of the nominal 30-mer with antibodies against the repair factor TFIIH which is bound to the oligomer or with antibodies against cyclobutane pyrimidine dimers (CPDs) 4 or (6 -4) photoproducts [(6 -4)PPs] and labeling of the excised oligonucleotide with either radioisotope or non-radioisotopic methods (7,8,13). The approach in its original form has been quite useful for characterizing DNA repair in mammalian cells in vivo (7,8,13) and also for mapping excision repair events genome-wide (14) but has so far only been used for studying repair of UVinduced DNA damage.…”

“…The method involves cell lysis followed by immunoprecipitation of the nominal 30-mer with antibodies against the repair factor TFIIH which is bound to the oligomer or with antibodies against cyclobutane pyrimidine dimers (CPDs) 4 or (6 -4) photoproducts [(6 -4)PPs] and labeling of the excised oligonucleotide with either radioisotope or non-radioisotopic methods (7,8,13). The approach in its original form has been quite useful for characterizing DNA repair in mammalian cells in vivo (7,8,13) and also for mapping excision repair events genome-wide (14) but has so far only been used for studying repair of UVinduced DNA damage. Because nucleotide excision repair operates on essentially all types of DNA lesions, in particular on the so-called "bulky DNA adducts" (4, 5), we suggested that with appropriate modifications the method could also be used for investigating other DNA damage repaired by nucleotide excision repair as well.…”

UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1)

“…Other genotoxic compounds were added directly to tissue culture media as indicated. Fractionation of cells to yield cytosolic and nuclear fractions was performed as previously described (33).…”

“…Radiolabeled oligonucleotides of known length were resolved on all gels as size markers. Excision repair activity was quantified using ImageQuant 5.2 software (GE Healthcare) as previously described (33).…”