Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

Abstract

In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1-and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4 M ammalian genomes are subjected substantial DNA damage from both endogenous processes [e.g., DNA replication, class switching recombination (CSR), etc.] and exogenous resources (e.g., ionic radiation, DNA-damaging chemicals, etc.). Evolutionarily conserved DNA repair pathways are essential to maintain both the structure integrity and the information accuracy of the genome (1). In eukaryotes, DNA double-strand breaks (DSBs), one of the most dangerous and severe types of DNA damage, are repaired mainly by two evolutionarily conserved repair pathways: homologous repair (HR) and nonhomologous end-joining (NHEJ) (2). NHEJ directly ligates two broken DSB ends, whereas HR uses a homologous template (in most of cases, the sister chromosome) for repair. Although NHEJ is simpler and faster than HR, repair by NHEJ often leads to change of sequences in the repair junctions via deletion, insertion, and mutations. Most importantly, NHEJ can also directly ligate two distant DSBs (both in cis-and transchromosomes), therefore leading to chromosomal deletions and translocations that are tightly linked to the genome evolution and carcinogenesis (3).The NHEJ in eukaryotes has been extensively studied in the last two decades (4-6). Mechanistically, NHEJ could be separated into several steps. First, in the DSB binding and tethering step, the DSB ends are recognized and bound by Ku70/Ku80 heterodimers that function as docking sites for other NHEJ factors. In the next end-processing step, nuclease and DNA polymerase activities are recruited to remove damaged or mismatched nucleotides and prepare the broken ends for ligation. Finally, DNA ligase IV (Lig4) complex, consisting of DNA Lig4 and its cofactor X-ray repair cross-complementing protein 4 (XRCC4), as well as the newly identified XRCC4-interacting factor (XLF), reseals the DSBs. Both V(D)J ...

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

“…Cell cycle regulation of template availability and processing may also affect the mechanism of telomere fusions (Escribano-Díaz et al 2013). We were able to sequence rare telomere-genomic fusions from LIG3 −/− :LIG4 −/− cells, reinforcing the notion of the coincidence of fusion foci with DNA replication, since LIG1 is the most rational protagonist mediating these LIG4-independent inter-chromosomal events (Arakawa et al 2012;Lu et al 2016). Further support arises from our finding of a conspicuous and significant association of LIG3 −/− :LIG4 −/− junctionproximal sequence with non-B DNA structures (Cooper et al 2011;Cer et al 2013) and a trend toward increased coincidence with fragile sites for LIG4 −/− junctions, implicating replication fork-stalling (Vissers et al 2009;Minca and Kowalski 2011;Ozeri-Galai et al 2011;Mizuno et al 2013) as a determinant of chromosome breakage and/or fusion.…”

“…This translated into 3.56-and 2.29-fold reductions in sequence-verified inter-chromosomal fusion junctions characterized in LIG4 −/− and LIG3 −/− :LIG4 −/− , respectively, compared with LIG3 −/− samples, indicating insufficient compensation for a nonredundant role of LIG4 in these long-range events. We consider that these observations provide evidence for a critical role of LIG4 in effecting inter-chromosomal telomere fusions, rather than indicating an inhibitory impact of LIG3, since we detected comparable increases in inter-chromosomal read frequencies and ratios to intra-chromosomal events for the LIG3 −/−:NC3 samples (with supra-normal levels of nuclear LIG3) as with the LIG3 , this provides important support for the presence of an additional ligase (ostensibly LIG1) (Arakawa et al 2012;Lu et al 2016) capable of mediating NHEJ in the absence of LIG3 and LIG4 or an alternative means of functional compensation of this DNA repair pathway in human cells.…”

“…In contrast, the alternative NHEJ (A-NHEJ) pathway involving both LIG3 and poly(ADP-ribose) polymerase 1 (PARP1) (Pascal and Ellenberger 2015) is associated with greater substrate resection to reveal microhomology that bridges and stabilizes junctions. More recently, DNA ligase 1 (LIG1) has been implicated as a functional substitute for LIG3 in chicken cells (Arakawa et al 2012) and a subset of LIG3 activities in mouse cells (Lu et al 2016). The interdependence or hierarchy of specialized LIG1 and LIG3 roles in A-NHEJ remains to be clarified in human cells.…”

Chromothripsis during telomere crisis is independent of NHEJ, and consistent with a replicative origin

“…Recent studies suggest telomere fusions are mediated by classical (C-NHEJ) and alternative (A-NHEJ) nonhomologous end joining (Smogorzewska et al 2002;Bunting and Nussenzweig 2013;Liddiard et al 2016). C-NHEJ typically involves blunt-end ligation by the XRCC4:LIG4 complex (Critchlow et al 1997;Walker et al 2001;Chang et al 2017), whereas A-NHEJ uses LIG3, with an auxiliary role for LIG1 (Simsek et al 2011;Lu et al 2016;Liddiard et al 2018). A-NHEJ additionally has a requirement for microhomology that can be exposed by preprocessing of the DNA end (Sfeir and Symington 2015; Chang et al 2017).…”

WRN regulates pathway choice between classical and alternative non-homologous end joining

“…During alt-NHEJ, MRE11 and PARP1 likely perform the DNA damage recognition, while CtIP and the MRN complex (MRE11, RAD50 and NBS1) process the broken ends by resection. Subsequently, the resected ends are ligated by DNA ligase I or ligase III (refs 19, 20, 21, 22, 23). …”