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The Minimal Bacillus subtilis Nonhomologous End Joining Repair Machinery  [PDF]
Miguel de Vega
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064232
Abstract: It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5′ end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3′ ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.
Nonhomologous DNA End Joining in Cell-Free Extracts  [PDF]
Sheetal Sharma,Sathees C. Raghavan
Journal of Nucleic Acids , 2010, DOI: 10.4061/2010/389129
Abstract: Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems. 1. Introduction Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks. Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Examples include base excision repair (base level changes), nucleotide excision repair (distortions in the DNA); and single-strand break repair. DSBs are considered as the most deleterious type of DNA damages, among different lesions. It can result in chromosomal rearrangements like translocations and cancer or cell death when unrepaired [1, 2]. DSBs can be generated by pathological or physiological agents. Pathological agents can be exogenous such as ionizing radiation, or chemotherapeutic agents like bleomycin (Figure 1). They can also be endogenous like oxidative free radicals, replication across a nick, inadvertent enzyme action at fragile sites (Figure 1), mechanical shearing at anaphase bridges, metabolic by products, and so forth [3–5]. Physiological processes such as V(D)J recombination, class switch recombination (CSR); and meiosis also introduce DSBs in our genome. Figure 1: Double-strand breaks (DSBs) are generated exogenously by ionizing radiation, or endogenously by free radicals or during V(D)J recombination in pre-B (bone marrow) and pre-T cells (thymus) by RAG complex or also during class switch recombination in activated B cells (in the peripheral lymphoid tissues such as spleen, lymph nodes, and Peyer’s patches). NHEJ involves the binding of Ku70 and Ku80 heterodimeric complex to the DNA ends, and DNA-PKcs in association with ARTEMIS. ARTEMIS is a - exonuclease in an unphosphorylated form while it is an endonuclease in a phosphorylated form. Artemis protein acts as an exonuclease and helps in resection of the ends. Polymerase family members are then recruited for DNA synthesis, which includes both template dependent and independent DNA synthesis. The resulting DNA ends are ligated by a specific DNA LIGASE IV
Positive selection on the nonhomologous end-joining factor Cernunnos-XLF in the human lineage
Adam Pavlicek, Jerzy Jurka
Biology Direct , 2006, DOI: 10.1186/1745-6150-1-15
Abstract: We obtained or reconstructed full-length coding sequences of chimpanzee, rhesus macaque, canine, and bovine Cernunnos-XLF orthologs from sequence databases and sequence trace archives. Comparison of coding sequences revealed an excess of nonsynonymous substitutions consistent with positive selection on Cernunnos-XLF in the human lineage. The hotspots of adaptive evolution are concentrated around a specific structural domain, whose analogue in the structurally similar XRCC4 protein is involved in binding of another nonhomologous end-joining factor, DNA ligase IV.Cernunnos-XLF is a microcephaly-associated locus newly identified to be under adaptive evolution in humans, and possibly played a role in human brain expansion. We speculate that Cernunnos-XLF may have contributed to the increased number of brain cells in humans by efficient double strand break repair, which helps to prevent frequent apoptosis of neuronal progenitors and aids mitotic cell cycle progression.This article was reviewed by Chris Ponting and Richard Emes (nominated by Chris Ponting), Kateryna Makova, Gáspár Jékely and Eugene V. Koonin.Reviewed by Chris Ponting and Richard Emes (nominated by Chris Ponting), Kateryna Makova, Gáspár Jékely and Eugene V. Koonin. For the full reviews, please go to the Reviewers' comments section.Double-strand breaks (DSBs) are highly cytotoxic DNA lesions caused by ionizing radiation, spontaneous chromosomal breaks, activity of cellular endonucleases, or during replication of other DNA lesions such as single-strand breaks. If unrepaired, DSBs efficiently trigger arrest of cell cycle progression and cell death by apoptosis [1]. In response to this danger, cells have developed mechanisms that repair DSBs. In eukaryotic cells, there are two major groups of DSB repair pathways [2]: homologous recombination (HR) and nonhomologous end-joining (NHEJ). In contrast to HR, NHEJ does not require a highly identical undamaged partner DNA strand to repair DSBs and, after some process
Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining  [PDF]
Cynthia L. Hendrickson,Shubhadeep Purkayastha,Elzbieta Pastwa,Ronald D. Neumann,Thomas A. Winters
Journal of Nucleic Acids , 2010, DOI: 10.4061/2010/823917
Abstract: In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA- to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA- appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA- protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways. 1. Introduction DNA double-strand breaks (DSBs) constitute the most cytotoxic form of DNA damage in the genome. DSBs are generated not only by exogenous sources, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors but also from endogenous cellular processes that generate reactive oxygen species [1, 2]. In mammalian cells, one of the major pathways for the repair of DSBs is nonhomologous end joining (NHEJ) [3, 4]. The principle proteins that participate in this DNA end joining pathway both in vitro [5–7] and in vivo [8–10] are the Ku 70/80 heterodimer, DNA-P , XRCC4, DNA ligase IV and XLF (XRCC4-like factor; also called Cernunnos) that has recently been identified as a binding partner of the DNA ligase IV-XRCC4 complex and as necessary for efficient ligation via NHEJ [11, 12]. Subsets of NHEJ may involve other factors such as Artemis [13]. Together with the Artemis protein, DNA-PKcs can stimulate processing of the DNA ends [14, 15]. Additional proteins, including DNA polymerases and , TDP1 (tyrosyl-DNA phosphodiesterase), PNK (polynucleotide kinase), and WRN (Werner’s syndrome helicase) are also likely to play a role in DSB repair [16]. Recent reports suggest that numerous other proteins, including ATM, histones H1 and H2AX, NBS1, and Mre11 may also have some influence on the NHEJ pathway [17–19]. NHEJ is a complex, multistep
Proofreading Activity of DNA Polymerase Pol2 Mediates 3′-End Processing during Nonhomologous End Joining in Yeast  [PDF]
Shun-Fu Tseng,Abram Gabriel,Shu-Chun Teng
PLOS Genetics , 2008, DOI: 10.1371/journal.pgen.1000060
Abstract: Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends, Pol2 is important for the recession of 3′ flaps that can form during imprecise pairing. Indeed, a mutation in the 3′-5′ exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3′ flaps. Thus, Pol2 performs a key 3′ end-processing step in NHEJ.
Structural Biology of DNA Repair: Spatial Organisation of the Multicomponent Complexes of Nonhomologous End Joining  [PDF]
Takashi Ochi,Bancinyane Lynn Sibanda,Qian Wu,Dimitri Y. Chirgadze,Victor M. Bolanos-Garcia,Tom L. Blundell
Journal of Nucleic Acids , 2010, DOI: 10.4061/2010/621695
Abstract: Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods—X-ray crystallography, electron microscopy and small angle X-ray scattering—can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere. 1. Introduction Nonhomologous End Joining (NHEJ) and Homologous Recombination (HR) comprise the two major modes of DNA double-strand break (DSB) repair in human cells. Although HR is dominant in late S/G2 phases when a sister chromatid is available [1], NHEJ, which does not require a template [2], plays a major role in G1/early S phase [1]. It is predicted that in humans about 50 endogenous DSBs per cell during each cell cycle may occur [3]. These are mainly generated by ionizing radiation, reactive oxygen species, and DNA replication across a nick [2]. Unrepaired DSBs can cause catastrophic gene loss during cell division, leading to chromosomal translocations, increased mutation rates, and carcinogenesis [4]. The NHEJ system is also responsible for programmed DSBs in V(D)J recombination [5] and class switch recombination [6] during development of immune diversity. NHEJ has an alternative end-joining pathway, which is mostly microhomology-mediated end joining [7] and is independent of NHEJ components [8]. Here NHEJ implies the main NHEJ pathway. The NHEJ pathway comprises three major steps: synapsis, end processing and ligation [9]. Synapsis is carried out by DNA-dependent protein kinase (DNA-PK) consisting of Ku70, Ku80, DNA-PK catalytic subunit (DNA-PKcs), and DNA. Ku70 and Ku80 form a ring-shaped heterodimer around the broken DNA ends and maintain them in proximity [10, 11]. DNA-PKcs, a very large protein belonging to the phosphatidylinositol-3-OH kinase (PI3K)-related kinase (PIKKs) family [12], is recruited through interaction
DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation  [PDF]
Deniz Simsek equal contributor,Erika Brunet equal contributor,Sunnie Yan-Wai Wong,Sachin Katyal,Yankun Gao,Peter J. McKinnon,Jacqueline Lou,Lei Zhang,James Li,Edward J. Rebar,Philip D. Gregory,Michael C. Holmes,Maria Jasin
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002080
Abstract: Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.
Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts  [PDF]
Natalie R. Gassman,Donna F. Stefanick,Padmini S. Kedar,Julie K. Horton,Samuel H. Wilson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049301
Abstract: Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.
De Novo CNV Formation in Mouse Embryonic Stem Cells Occurs in the Absence of Xrcc4-Dependent Nonhomologous End Joining  [PDF]
Martin F. Arlt,Sountharia Rajendran,Shanda R. Birkeland,Thomas E. Wilson ,Thomas W. Glover
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002981
Abstract: Spontaneous copy number variant (CNV) mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and de novo pathogenic CNVs, features recapitulated in experimental systems in which CNVs are induced by exogenous replication stress. To test whether the canonical nonhomologous end joining (NHEJ) pathway of double-strand break (DSB) repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ–deficient Xrcc4?/? mouse embryonic stem (ES) cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4?/? cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.
Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks  [PDF]
Swetha Parvathaneni, Alexei Stortchevoi, Joshua A. Sommers, Robert M. Brosh, Sudha Sharma
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062481
Abstract: Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.
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