Base damage immediately upstream from double-strand break ends is a more severe impediment to nonhomologous end joining than blocked 3′-termini

Abstract Radiation-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions that are typically repaired by nonhomologous end joining (NHEJ) in human cells. Our previous work indicated that the highly cytotoxic DSBs formed by 125I decay possess base damage clustered within 8 to 10 bases of the break and 3′-phosphate (P) and 3′-OH ends. This study examined the effect of such structures on NHEJ in in vitro assays employing either 125I decay-induced DSB linearized plasmid DNA or structurally defined duplex oligonucleotides. Duplex oligonucleotides that possess either a 3′-P or 3′-phosphoglycolate (PG) or a ligatable 3′-OH end with either an AP site or an 8-oxo-dG 1 nucleotide upstream (−1n) from the 3′-terminus have been examined for reparability. Moderate to severe end-joining inhibition was observed for modified DSB ends or 8-oxo-dG upstream from a 3′-OH end. In contrast, abolition of end joining was observed with duplexes possessing an AP site upstream from a ligatable 3′-OH end or for a lesi...