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 indicates 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 examines 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 ligateable 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 ligateable 3′-OH end, or for a lesion combination involving 3′-P plus an upstream 8-oxo-dG. In addition, base mismatches at the -1n position are also strong inhibitors of NHEJ in this system, suggesting that destabilization of the DSB terminus as a result of base loss or improper base pairing may play a role in the inhibitory effects of these structures. Furthermore, we provide data indicating that DSB end joining is likely to occur prior to removal or repair of base lesions proximal to the DSB terminus. Our results show that base damage or base loss near a DSB end may be a severe block to NHEJ, and that complex combinations of lesions presented in the context of a DSB may be more inhibitory than the individual lesions alone. In contrast, blocked DSB 3′-ends alone, are only modestly inhibitory to NHEJ. Finally, DNA ligase activity is implicated as being responsible for these effects.

Spectrum of Radiation-Induced Clustered Non-DSB Damage – A Monte Carlo Track Structure Modeling and Calculations

“…This kind of analysis is particularly important because the majority of DSBs contain at least one base damage, and if included in the complexity index of the DSB, this would suggest that most (23,69). The simulations carried out in this study are capable of determining the exact position of base damages relative to strand breaks.…”

“…These findings have now been extended to include a slow component in base excision repair (BER), a result of closely spaced base lesions and SSBs (15). This finding is important, since non-DSB lesions can also contribute to deleterious effects, such as mutagenesis and cytotoxicity (20)(21)(22)(23).…”

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells

“…chemotherapy or ionizing radiations) can lead to such 3Ј-DNA blocking lesions. Ionizing radiation (IR) induces a variety of DNA lesions, including a small number of lethal DSBs and many more single strand breaks with 3Ј-PG ends (40,41). Bleomycin induces DSBs with 3Ј-PG ends (42).…”

Specificity of the dRP/AP Lyase of Ku Promotes Nonhomologous End Joining (NHEJ) Fidelity at Damaged Ends

“…Abasic sites are associated with chromosome breaks generated by exposure to ionizing radiation (as well as other sources of reactive oxygen species) (5, 10) and aborted base excision repair (BER) (11)(12)(13)(14) and associated with intermediates in immunoglobulin isotype class switch recombination (15,16). Such lesions are a strong block to ligation when present near a DSB terminus and must be excised (7)(8)(9)12). Abasic sites in this context can be specifically excised by a 5Ј-dRP/AP lyase activity that is part of the classically defined NHEJ pathway (9), similar to a step mediated by DNA polymerase ␤ in base excision repair (BER)/singlestand break repair (17)(18)(19)(20).…”