DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining

Abstract

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/ XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency. (Cancer Res 2005; 65(10): 4020-30)

Harnessing the complexity of DNA-damage response pathways to improve cancer treatment outcomes

“…Interestingly, PARP has also been implicated in a slow alternative NHEJ pathway that is separate from the DNA-PK-dependent classical NHEJ (Audebert et al, 2004(Audebert et al, , 2008Wang et al, 2005Wang et al, , 2006Shrivastav et al, 2008;Terzoudi et al, 2008;Iliakis, 2009;Robert et al, 2009). This finding may further justify the use of PARPi in HR-deficient cancers, especially since the alternative NHEJ pathway may be enhanced in G2 (Wu et al, 2008).…”

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

“…The fact that both g-H2AX and PFGE analysis showed a decrease in DSB frequency in such situation (see Figure 5) means that XRCC4 alternative pathways can process DSBs in S phase. Such pathways could be NHEJ pathways alternative to XRCC4 (Wang et al, 2003(Wang et al, , 2005Audebert et al, 2004) and/or HR, which is highly efficient in S phase. The latter hypothesis is supported by the observation that the frequency of Rad51 focipositive cells was higher with XRCC4 À cells irradiated in G1 compared to G2, whereas this was not the case with complemented cells (see Figure 2b).…”

DNA double-strand break repair and development

“…For instance, DNA ligase III (Lig3) has been implicated as a alternate ligase that may function in NHEJ (Wang et al, 2005a;Windhofer et al, 2007). In vitro evidence implicating PARP-1 and Xrcc1/Lig3 in NHEJ has also been obtained through end-joining assays (Audebert et al, 2004).…”