title: Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3'-phosphoglycolate termini in SCAN1 cells.
abstract: To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3'-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3'-phosphate and 3'-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3'-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3'-PG and 3'-phosphate termini on 1-base 3' overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3'-PG termini were processed more slowly than 3'-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3'-PG blocking groups for DSB repair. DSBs with 3'-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3'-PG ends.
authors.affiliation: Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA and Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Akopiants
authors.forename: Susovan
authors.initials: S
authors.lastname: Mohapatra
authors.forename: Vijay
authors.initials: V
authors.lastname: Menon
authors.forename: Tong
authors.initials: T
authors.lastname: Zhou
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Valerie
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Povirk
mesh_terms: D002461:Cell Line, Transformed; D004247:DNA / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D051747:DNA-Activated Protein Kinase / Q000172:deficiency; D006016:Glycolates / Q000032:analysis*; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020754:Spinocerebellar Ataxias / Q000235:genetics; D009353:Zinostatin / Q000633:toxicity
chemical_list: D006016:Glycolates; D004247:DNA; D009353:Zinostatin; D051747:DNA-Activated Protein Kinase; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C013193:phosphoglycolate