Absence Of Nonhomologous End Joining Research Articles

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo etal. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Dual loss of human POLQ and LIG4 abolishes random integration

Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.

Impaired Resection of Meiotic Double-Strand Breaks Channels Repair to Nonhomologous End Joining in Caenorhabditis elegans

Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. Mre11 is one member of the MRX/N (Mre11, Rad50, and Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in Saccharomyces cerevisiae. In Caenorhabditis elegans, evidence for the MRX/N role in DSB resection is limited. We report the first separation-of-function allele, mre-11(iow1) in C. elegans, which is specifically defective in meiotic DSB resection but not in formation. The mre-11(iow1) mutants displayed chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation was greatly reduced in mre-11(iow1) mutants. Irradiation-induced DSBs during meiosis failed to be repaired from early to middle prophase I in mre-11(iow1) mutants. In the absence of a functional HR, our data suggest that some DSBs in mre-11(iow1) mutants are repaired by the nonhomologous end joining (NHEJ) pathway, as removing NHEJ partially suppressed the meiotic defects shown by mre-11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to EXO-1-dependent HR repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in middle meiotic prophase I and in blocking NHEJ.

Direct and inverted repeats elicit genetic instability by both exploiting and eluding DNA double-strand break repair systems in mycobacteria.

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.

A Robust Network of Double-Strand Break Repair Pathways Governs Genome Integrity during C. elegans Development

To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ) or single-strand annealing (SSA). Here, we created a transgenic reporter system in C. elegans to investigate the relative contribution of these pathways in somatic cells during animal development. Although all three canonical pathways contribute to repair in the soma, in their combined absence, animals develop without growth delay and chromosomal breaks are still efficiently repaired. This residual repair, which we call alternative end-joining, dominates DSB repair only in the absence of NHEJ and resembles SSA, but acts independent of the SSA nuclease XPF and repair proteins from other pathways. The dynamic interplay between repair pathways might be developmentally regulated, because it was lost from terminally differentiated cells in adult animals. Our results demonstrate profound versatility in DSB repair pathways for somatic cells of C. elegans, which are thus extremely fit to deal with chromosomal breaks.

Cyclin A/CDK2 Regulates V(D)J Recombination by Coordinating RAG-2 Accumulation and DNA Repair

Accumulation of the V(D)J recombinase protein RAG-2 is restricted to G0/G1 cells by phosphorylation-mediated degradation at the G1-S boundary. Here cyclin A/CDK2 is shown to oppose RAG-2 accumulation; conversely, RAG-2 is induced by p27 Kip1 and related CDK inhibitors. Coinduction of RAG-2 and G1 delay by p27 Kip1 is accompanied by strong stimulation of V(D)J recombination. Unexpectedly, induction of RAG-2 accumulation in the absence of G1 delay has no effect on recombination frequency. p27 Kip1 may stimulate V(D)J recombination by coordinating accumulation of RAG-2 with prolongation of G1, when nonhomologous end joining is preferentially active. Consistent with this, enforced expression of RAG-2 throughout cell cycle is associated with accumulation of aberrant recombination products reminiscent of those formed in the absence of nonhomologous end joining.