Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

Abstract

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3′ terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.

Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

“…On the other hand, it seems unlikely that the residual 3′-dephosphorylation is the result of general broad-specificity phosphatases, since it is regulated by DNA-PK. Furthermore, it appears to act specifically on 3′- and not 5′-phosphates, as indicated by the retention of 5′- 32 P label on DSB substrates even after incubation in whole-cell extracts for many hours [14,47]. Rather, overall the data suggest an enzyme that acts specifically in NHEJ, in a manner similar to PNKP but less efficiently.…”

XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway

“…Using blunt-ended DSB substrates harboring the oxidatively modified nonplanar base thymine glycol (Tg) at the first, second, third or fifth positions from one 3′ terminus (Tg1, Tg2, Tg3 and Tg5 respectively), NHEJ of damaged DSB ends was examined in human whole-cell extracts [38]. In XLF-deficient extracts, end joining of all substrates, including unmodified blunt ends, was completely dependent on addition of recombinant XLF.…”

Complex genetic interactions between DNA polymerase β and the NHEJ ligase

“…5, however, POLB À/À cells were slightly but reproducibly more resistant to highdose x-ray (>4 Gy) than WT cells. Consistent with this view, Almohaini et al [35] have reported that BER can interfere with NHEJ when thymine glycol is formed at the fifth base from the 3' terminus of one DNA end. As x-ray can induce DSB-clustered lesions, which associate DSBs and several base damage and abasic sites in close vicinity [34], POLb-dependent BER may prevent efficient repair of such lesions in the presence of NHEJ.…”

“…As x-ray can induce DSB-clustered lesions, which associate DSBs and several base damage and abasic sites in close vicinity [34], POLb-dependent BER may prevent efficient repair of such lesions in the presence of NHEJ. Consistent with this view, Almohaini et al [35] have reported that BER can interfere with NHEJ when thymine glycol is formed at the fifth base from the 3' terminus of one DNA end. Live cell imaging also revealed that BER factors (such as PARP-1 and XRCC1) rapidly accumulate at laser-induced DNA damage sites, in a manner similar to NHEJ factors [36].…”