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(PP295) Upstream lesions are a more severe impediment to non-homologous end joining than blocked 3′-ends.
Datta, Kamal*,1, Neumann, Ronald1, Winters, Thomas1, 1 Nuclear Medicine Department/ Warren Grant Magnuson Clinical Center, Bethesda, MD
ABSTRACT- DNA double-strand breaks (DSBs) are implicated as a critical cytotoxic lesion produced by radiation. Radiation-induced DSB complexity increases with increasing linear energy transfer (LET), and high-LET radiation-induced DSBs are known to pose a significant challenge to the cellular repair machinery. The objective of the study is to investigate the implication of 3′ blocked ends versus upstream damage on DSB repair by non-homologous end joining (NHEJ). Hela whole cell extract was used for the NHEJ assay. Ku-70, DNA-Pkcs and Ligase IV antibody was used for gel supershift and Ku-70 also for immunodepletion. Our previous observations have shown that 125-I-mediated high-LET-like DSBs contain clustered damage within 8 to 10 base of the break site. We have also shown that unlike low-LET breaks, these DSBs mostly possess phosphate and OH but no phosphoglycolate at the 3′-end. DSBs with such end-structures are not expected to be refractory to repair; and yet, 125-I-induced DSBs were poorly repaired by HeLa whole-cell extracts in in vitro assays. Even when 3′- phosphates (3′-P) were removed by T4 PNK phosphatase activity and all four dNTPs were added, 125-I-DSBs remained refractory to repair. Hence, we hypothesized that upstream damage rather than blocking end groups could be an important determinant for DSB repair. To test this hypothesis we synthesized a 75 bp duplex oligonucleotide with a 4 base 5′-overhang at one end and the other being blunt. The overhang 5′-end was phosphorylated with radioactive 32-P thus allowing joining at this end. HeLa whole-cell extract end-joined this substrate and produce dimer products. Gel supershift assays indicate that the oligo duplex binds Ku-70, DNA-PKcs, and DNA ligase IV, suggesting dimer formation to be mediated by NHEJ. Ku-70 immunodepleted HeLa extract exhibited decreased product yield proportional to the reduction of Ku-70 protein, further supporting a NHEJ repair mechanism. Substitution of the undamaged duplex oligo with oligos possessing either a 3′-P blocked end, or an AP-site 2 bases upstream from a ligateable 3′-OH end, results in moderately reduced end-joining and abolition of end-joining respectively independent of dNTPs. Our results indicate that abasic sites upstream from DSB ends are severe blocks to NHEJ, whereas blocked 3′-ends are only modestly inhibitory.
Key words: DSB, repair, radiation, NHEJ
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