Alternative End-joining Repair Research Articles

Targeting FEN1 Suppresses the Proliferation of Chronic Myeloid Leukemia Cells Through Regulating Alternative End-Joining Pathways.

Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.

Patterns of Nucleotide Deletion and Insertion Inferred from Bacterial Pseudogenes.

Pseudogenes are a paradigm of neutral evolution and their study has the potential to reveal intrinsic mutational biases. However, this potential is mitigated by the fact that pseudogenes are quickly purged from bacterial genomes. Here, we assembled a large set of pseudogenes from genomes experiencing reductive evolution as well as functional references for which we could establish reliable phylogenetic relationships. Using this unique dataset, we identified 857 independent insertion and deletion mutations and discover a pervasive bias towards deletions, but not insertions, with sizes multiples of 3 nt. We further show that selective constraints for the preservation of gene frame are unlikely to account for the observed mutational bias and propose that a mechanistic bias in alternative end-joining repair, a recombination-independent double strand break DNA repair mechanism, is responsible for the accumulation of 3n deletions.

The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.

Recovery of Alternative End-Joining Repair Products From Drosophila Embryos.

In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ.

Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining.

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3’ overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.

Linking DNA polymerase theta structure and function in health and disease.

DNA polymerase theta (Pol θ) is an error-prone A-family polymerase that is highly conserved among multicellular eukaryotes and plays multiple roles in DNA repair and the regulation of genome integrity. Studies conducted in several model organisms have shown that Pol θ can be utilized during DNA interstrand crosslink repair and during alternative end-joining repair of double-strand breaks. Recent genetic and biochemical studies have begun to elucidate the unique structural features of Pol θ that promote alternative end-joining repair. Importantly, Pol θ-dependent end joining appears to be important for overall genome stability, as it affects chromosome translocation formation in murine and human cell lines. Pol θ has also been suggested to act as a modifier of replication timing in human cells, though the mechanism of action remains unknown. Pol θ is highly upregulated in a number of human cancer types, which could indicate that mutagenic Pol θ-dependent end joining is used during cancer cell proliferation. Here, we review the various roles of Pol θ across species and discuss how these roles may be relevant to cancer therapy.

Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches.

Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase θ, and reduction of its activity in mammalian cells increases sensitivity to double-strand break inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ employs a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction to the 3’-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining.

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.

Loss of the bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila

BackgroundThe BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, we used a lacZ reporter system in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan.ResultsOur data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues.ConclusionsThese results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan.

Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.