DOCSLIB.ORG

Alternative Endings

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Alternative Endings COMMENTARY Alternative endings James E. Haber* Department of Biology and Rosenstiel Center, Brandeis University, Waltham, MA 02454-9110 he repair of chromosome breaks is essential to maintain genome integrity. Both in mammalian cells and in budding yeast, repair Tin the G1 phase of the cell cycle is particu- larly dependent on nonhomologous end-joining (NHEJ) to repair broken chromosomes. NHEJ is important in re- pairing double-strand breaks (DSBs) that arise during both V(D)J recombination and class switch recombination (CSR) events in the immune system. End-joining also is responsible for generating chromo- somal translocations that are associated with various cancers. Work by Guirouilh- Barbat et al. (1) published in a recent issue of PNAS and by several other labo- ratories (2–4) has revealed that–in addition to the ‘‘classical’’ NHEJ path- way–there is a robust alternative pathway that also contributes to the generation of translocations. Our current understanding of the mechanisms of NHEJ results from a dynamic interplay between genetics and biochemistry both in mammals and in budding yeast (for reviews, see refs. 5–7). A major motivation to identify Fig. 1. Induction of NHEJ by I-SceI endonuclease cleavage. I-SceI cleaves within an 18-bp nonsymmetric the genes responsible for NHEJ has site to generate 4-bp, 3Ј overhanging ends. Joining of the CMV promoter to the coding sequence of the been the study of end-joinings in both CD4 gene creates CD4ϩ cells that can be scored by flow cytometry. Inversion of one of the cleavage sites V(D)J recombination and Ig CSR in creates partially complementary ends. Fully complementary ends are generally joined by perfect ligation mammals, driven by the identification when the ‘‘classical’’ NHEJ apparatus (including Ku proteins, LIG4, and XRCC4) is functional. With partially of immune-compromised humans. A compatible ends, NHEJ still permits ligation even though two sites (underlined) are mispaired. Among second impetus has been the iden- other joinings of the overhanging ends are 2-bp deletions requiring removal of 2 nt from each end before tification of genes conferring x-ray they can be ligated. In the absence of Ku proteins or XRCC4, most ends are joined by the alternative NHEJ resistance. These studies led to the pathway by using MMEJ. identification of DNA-protein kinase catalytic subunit (PKcs) and the Ku70 die of lymphomas characterized by budding yeast in the case when the and Ku80 proteins, which play a cen- gene fusions between the Ig heavy- 4-bp protruding chromosome ends, cre- tral role both in immune cell recombi- chain IgH locus and c-Myc; they also ated by a site-specific nuclease, were nation and in resistance to ionizing show evidence of many other nonrecip- completely nonhomologous (14). radiation. Further work led to the rocal translocations. Surprisingly, the Whereas NHEJ between ends that had identification of a number of XRCC translocation junctions still appear to even partial complementarity were all genes conferring radio-resistance. Ku-dependent, end-joinings between Subsequent studies in budding yeast be the result of nonhomologous end noncomplementary ends were efficient confirmed that Ku proteins were also fusions rather than of homologous but Ku-independent. MMEJ must in- essential for NHEJ (there is no DNA- recombination between repeated se- PKcs). Research using budding yeast quences. Thus, there had to be an al- ternative NHEJ process not requiring identified other NHEJ components, Author contributions: J.E.H. wrote the paper. notably DNA ligase 4, its partner LIG4 or XRCC4 and probably also not The author declares no conflict of interest. Lif1 (whose mammalian homolog is requiring the Ku proteins. A key fea- ture of this pathway is that joinings See companion article on page 20902 in issue 52 of volume XRCC4), and Nej1 (whose mammalian 104. homolog is XLF/Cernunnos)†. Ku- occur preferentially between sequences *E-mail: [email protected]. deficient mice are viable but severely near the ends that shared as much as 7 nt of homology (12). Similarly, end- †The identification of yeast Nej1 and mammalian XLF/Cer- immunocompromised, whereas mice nunnos is a fascinating example of many possible strate- lacking DNA ligase 4 (LIG4) and joining of blunt-ended linearized plas- gies to identify the same gene. NEJ1 was found simulta- XRCC4 die embryonically; however, mids in cells without KU80 involve the neously by five groups using four different approaches, they can be rescued by also knocking resection and annealing of short ho- including gene-expression profiling, two-hybrid interac- tions, and screenings of libraries of yeast gene deletions. out the p53-mediated apoptotic path- mologous sequences rather than simply XLF/Cernunnos was identified both by complementation way that eliminates cells with unre- joining the blunt ends (13). An analo- of a human immune-deficient mutation and by two-hybrid paired DSBs (8–11). However, these gous microhomology-mediated end- analysis. immune-deficient mice almost always joining (MMEJ) pathway was seen in © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711334105 PNAS ͉ January 15, 2008 ͉ vol. 105 ͉ no. 2 ͉ 405–406 Downloaded by guest on September 27, 2021 volve significant 5Ј–3Ј resection of the backup NHEJ system operating without predominates when it is functional. DNA ends to expose regions with 2- to 10-bp XRCC4 (and presumably LIG4). Most of ligase 3, with its associated XRCC1 pro- microhomologies. Yeast MMEJ also the junctions were deletions with micro- tein, is a candidate for the ligase that sub- needs the Rad1-Rad10 endonuclease homologies at the junction. stitutes for LIG4 (16). It has also been apparently to trim the 3Ј ends Yan et al. (3) examined the end-joinings suggested that PARP is important (17, extending beyond the annealed associated with Ig IgH CSR in mouse B 18). The filling-in of small gaps created microhomologies. cells. The absence of LIG4 or XRCC4 during repair of noncompatible ends also A systematic study of alternative path- reduces CSR substantially, but there were requires DNA polymerases ␭ or ␮ (19, ways of NHEJ in mammalian cells was still many switches, most of which appear 20). Whether the Rad1–Rad10 homologs carried out by Lopez and coworkers (15) to have used microhomologies. Moreover, XPF-ERCC1 play a similar role in remov- Ј by using I-SceI site-specific endonuclease there were still many chromosomal trans- ing the extremities of the 3 tails after to create multiple chromosomal DSBs microhomology mediated annealing, as in whose joinings could be scored by flow yeast, is not known; an intriguing clue cytometry. They used I-SceI cleavage sites Ku-deficient mice are comes from the fact that this complex is arranged so that the protruding 4Ј-bp 3Ј required in the fusions of telomere se- ends were either fully complementary or viable but severely quences that have partially cohesive ends (when one site was inverted) partially (21). Yeast MMEJ also requires the complementary. With fully complemen- immunocompromised. Mre11–Rad50 complex (14); it will be tary ends, most end-joinings were perfect interesting to see whether these proteins ligations, restoring the cleavage site. Par- are also required in mammalian cells. It has been known for a decade that tially complementary ends also annealed locations between the IgH locus and other Ku-defective mice are not as deficient in despite mismatches within the junction, chromosomes. Thus, there must be a V(D)J recombination and are more via- sometimes yielding small deletions or in- ‘‘nonclassical’’ NHEJ mechanism that acts ble than XRCC4- or LIG4-defective ani- sertions caused by misalignments (Fig. 1). when LIG4/XRCC4 is absent. In a paral- mals. This observation might lead one Deleting KU80 reduced NHEJ with com- lel study, de Villartay and coworkers (4) to the conclusion that there were plementary ends but surprisingly had little used a conditional XRCC4 knockout mu- XRCC4-dependent mechanisms that effect on the partially cohesive ends; how- tation to show that an alternative NHEJ acted independent of Ku, but in fact it ever, many of the joints showed mis- pathway appeared to be used in CSR. Fi- now looks as if this interpretation is in- aligned ends or larger deletions, with nally, Corneo et al. (2) made the surpris- correct. Instead it seems that Ku plays junctions containing short regions of mi- ing discovery that alternative NHEJ can an XRCC4-independent role in regulat- crohomology, as in yeast MMEJ. be elicited in cells wild type for all general ing NHEJ, apparently in preventing the In the aforementioned article in PNAS NHEJ factors. V(D)J recombination is alternative MMEJ machinery from gain- by Guirouilh-Barbat et al. (1) and in sev- initiated by RAG1/RAG2 proteins that ing access to the DSB ends when KU70/ eral recent papers (2–4), the characteriza- not only create the DSBs but also hold KU80 are associated with the ends. In tion of an alternative, Ku-independent and protect the ends. In Rag proteins de- budding yeast, deletion of Ku proteins MMEJ mechanism in mammalian cells leted for certain regions, recombination is allows more robust 5Ј–3Ј resection of has been shown to be independent of efficient, but the outcomes are skewed the ends, which is consistent with the LIG4 and XRCC4. In the article by toward MMEJ events. Alternative NHEJ idea that Ku acts to shelter ends from Guirouilh-Barbat et al., joinings of I-SceI- activity can be found even with wild-type degradation. Resection of the ends is induced DSBs were examined in XRCC4- Rag proteins. These studies make clear required for MMEJ. Indeed, the finding deficient cells. Both with fully or partially that MMEJ is an important process in that Ku deficiency rescues the lethality cohesive ends, there was a Ͼ5-fold reduc- preserving overall genome integrity.
Load more

Recommended publications
  • Plugged Into the Ku-DNA Hub: the NHEJ Network Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou
    Plugged into the Ku-DNA hub: The NHEJ network Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou To cite this version: Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou. Plugged into the Ku-DNA hub: The NHEJ network. Progress in Biophysics and Molecular Biology, Elsevier, 2019, 147, pp.62-76. 10.1016/j.pbiomolbio.2019.03.001. hal-02144114 HAL Id: hal-02144114 https://hal.archives-ouvertes.fr/hal-02144114 Submitted on 29 May 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Progress in Biophysics and Molecular Biology xxx (xxxx) xxx Contents lists available at ScienceDirect Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio Plugged into the Ku-DNA hub: The NHEJ network * Philippe Frit a, b, Virginie Ropars c, Mauro Modesti d, e, Jean Baptiste Charbonnier c, , ** Patrick Calsou a, b, a Institut de Pharmacologie et Biologie Structurale, IPBS, Universite de Toulouse, CNRS, UPS, Toulouse, France b Equipe Labellisee Ligue Contre
    [Show full text]
  • DNA Ligase IV Syndrome; a Review Thomas Altmann1 and Andrew R
    Altmann and Gennery Orphanet Journal of Rare Diseases (2016) 11:137 DOI 10.1186/s13023-016-0520-1 REVIEW Open Access DNA ligase IV syndrome; a review Thomas Altmann1 and Andrew R. Gennery1,2* Abstract DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation. Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development. Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases. Keywords: DNA Ligase 4, Severe combined immunodeficiency, Primordial dwarfism, Radiosensitive, Lymphoid malignancy Background factors include intracellular events such as DNA replica- DNA ligase IV deficiency (OMIM 606593) or LIG4 syn- tion and meiosis, and extracellular events including drome (ORPHA99812), also known as Ligase 4 syn- damage by reactive oxygen species and ionising radi- drome, is a rare autosomal recessive disorder ation.
    [Show full text]
  • Cr2007108.Pdf
    npg Michael R Lieber et al. Cell Research (2008) 18:125-133. npg125 © 2008 IBCB, SIBS, CAS All rights reserved 1001-0602/08 $ 30.00 REVIEW www.nature.com/cr Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non- homologous DNA end joining: relevance to cancer, aging, and the immune system Michael R Lieber1, Haihui Lu1, Jiafeng Gu1, Klaus Schwarz2 1USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Rm 5428, Los Angeles, CA 90089-9176, USA; 2Institute for Clinical Transfusion Medicine & Immunogenetics, Institute for Transfusion Medicine, Univer- sity Hospital, Ulm, Germany Nonhomologous DNA end joining (NHEJ) is the primary pathway for repair of double-strand DNA breaks in hu- man cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect ad- ditional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configura- tions reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual com- ponents also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost. Keywords: nonhomologous DNA end joining (NHEJ), Ku, DNA-PKcs, Artemis, Cernunnos/XLF, ligase IV, XRCC4, poly- merase µ, polymerase λ Cell Research (2008) 18:125-133.
    [Show full text]
  • Mouse Lig4 Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Lig4 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Lig4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Lig4 gene (NCBI Reference Sequence: NM_176953 ; Ensembl: ENSMUSG00000049717 ) is located on Mouse chromosome 8. 2 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000095476). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Lig4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-191P3 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Null homozygotes die late in gestation with extensive CNS apoptosis, blocked lymphopoeiesis and failure of V(D)J joining. Carrier fibroblasts show elevated chromosome breaks. ~40% of homozygous hypomorphs survive, with retarded growth, reduced PBL and progressive loss of hematopoietic stem cells. Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of intron 1 for 5'-loxP site insertion: 2223 bp. The size of effective cKO region: ~3006 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele T A 5' gRNA region G 3' 1 2 Targeting vector T A G Targeted allele T A G Constitutive KO allele (After Cre recombination) Legends Exon of mouse Lig4 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • (XRCC1) Deficiency Enhances Class Switch Recombination and Is
    X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining Li Han1, Weifeng Mao1,2, and Kefei Yu3 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 Edited* by Frederick W. Alt, Howard Hughes Medical Institute, Harvard Medical School, Children’s Hospital, Immune Disease Institute, Boston, MA, and approved February 10, 2012 (received for review December 15, 2011) DNA double-strand breaks (DSBs) are essential intermediates in Ig the DNA end, the DNA-dependent protein kinase (DNA-PKcs) gene rearrangements: V(D)J and class switch recombination (CSR). that regulates end joining by phosphorylating other proteins (in- In contrast to V(D)J recombination, which is almost exclusively de- cluding itself), and the ligase complex containing XLF, XRCC4, pendent on nonhomologous end joining (NHEJ), CSR can occur in and DNA ligase 4. Also involved is a growing list of auxiliary NHEJ-deficient cells via a poorly understand backup pathway (or factors, including end processing nucleases (e.g., Artemis) and pathways) often termed alternative end joining (A-EJ). Recently, polymerases (μ and λ), polynucleotide kinases, 53BP1, and many several components of the single-strand DNA break (SSB) repair DNA damage response proteins (ATM, H2AX, Chk1, etc.). machinery, including XRCC1, have been implicated in A-EJ. To de- Although both V(D)J and class switch recombination rely on termine its role in A-EJ and CSR, Xrcc1 was deleted by targeted the generation and repair of DSBs, the dependence on NHEJ mutation in the CSR proficient mouse B-cell line, CH12F3.
    [Show full text]
  • Polymerase Δ Promotes Chromosomal Rearrangements and Imprecise Double-Strand Break Repair
    Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair Jacob V. Layera, Lydie Debaizea, Alexandria Van Scoyka, Nealia C. Houseb, Alexander J. Brownc, Yunpeng Liud, Kristen E. Stevensone, Michael Hemannd, Steven A. Robertsc, Brendan D. Priceb, David M. Weinstocka,f,g,1, and Tovah A. Dayh,1 aDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; bDepartment of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; cSchool of Molecular Biosciences, Washington State University, Pullman, WA 99164; dThe Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139; eDepartment of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02215; fCancer Biology Program, Broad Institute of MIT and Harvard University, Cambridge, MA 02142; gBiological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02215; and hDepartment of Biology, Northeastern University, Boston, MA 02115 Edited by James E. Haber, Brandeis University, Waltham, MA, and approved September 9, 2020 (received for review July 10, 2020) Recent studies have implicated DNA polymerases θ (Pol θ) and β and λ, can also be recruited for end-resection and gap-filling (9, (Pol β) as mediators of alternative nonhomologous end-joining 10). The XRCC4/LIGIV complex is recruited and ligates both (Alt-NHEJ) events, including chromosomal translocations. Here strands (11). we identify subunits of the replicative DNA polymerase δ (Pol δ) The third type of repair, alternative NHEJ (Alt-NHEJ), is as promoters of Alt-NHEJ that results in more extensive intrachro- often described as a back-up end-joining process, as it resolves a mosomal mutations at a single double-strand break (DSB) and greater fraction of DSBs when C-NHEJ is compromised (12).
    [Show full text]
  • Rabbit Anti-Human LIG4 (Phospho-Thr650) Polyclonal Antibody (CABT-L3791) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
    Rabbit Anti-Human LIG4 (Phospho-Thr650) polyclonal antibody (CABT-L3791) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Product Overview Rabbit Anti-Human HDAC5 (Phospho-Ser498) polyclonal antibody. This antibody detects endogenous levels of DNA Ligase 4 only when phosphorylated at Thr650. Specificity Target Modification: Phospho. Modification Sites: Human: T650 Target Human DNA Ligase 4 (Phospho-Thr650) Immunogen The antiserum was produced against synthesized peptide derived from human DNA Ligase 4 around the phosphorylation site of Thr650. Immunogen range: 616-665 Isotype IgG Source/Host Rabbit Species Reactivity Human Purification Affinity Purified Conjugate Unconjugated Applications WB, IHC, ELISA Molecular Weight 103 kDa Preparation The antibody was purified from rabbit antiserum by affinity-chromatography using phospho peptide. The antibody against non-phospho peptide was removed by chromatography using corresponding non-phospho peptide. Format Liquid Concentration Lot specific Buffer Rabbit IgG in PBS (without Mg2+ and Ca2+), pH 7.4, 150mM NaCl and 50% glycerol. Preservative 0.02% Sodium Azide Storage Stable at -20°C for at least 1 year. Ship Wet ice Warnings For research use only. 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved BACKGROUND Introduction The protein encoded by this gene is a DNA ligase that joins single-strand breaks in a double- stranded polydeoxynucleotide in an ATP-dependent reaction. This protein is essential for V(D)J recombination and DNA double-strand break (DSB) repair through nonhomologous end joining (NHEJ).
    [Show full text]
  • Sister Chromatid, but Not NHEJ-Mediated Inter-Chromosomal Telomere
    Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Sister chromatid, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4 Kate Liddiard,1 Brian Ruis,2 Taylor Takasugi,2 Adam Harvey,2 Kevin E. Ashelford, 1 Eric A. Hendrickson2¶ and Duncan M. Baird1¶ ¶ Joint senior authors 1Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, United Kingdom. 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA. Corresponding author: Duncan Baird, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK. Email: [email protected] Running title: A- and C-NHEJ of dysfunctional human telomeres Keywords: Telomere, genome instability, DNA repair, inter-chromosomal, intra- chromosomal, non-homologous end-joining (NHEJ), DNA ligase, microhomology, crisis, double-stranded DNA break (DSB) 1 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Telomeres shorten with each cell division and can ultimately become substrates for non-homologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterised over 1400 telomere fusion events at the single-molecule level, using a combination of high- throughput sequence analysis together with experimentally-induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse non-telomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions.
    [Show full text]
  • Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III
    Genes 2015, 6, 385-398; doi:10.3390/genes6020385 OPEN ACCESS genes ISSN 2073-4425 www.mdpi.com/journal/genes Review Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III Hiroshi Arakawa 1,* and George Iliakis 2 1 IFOM-FIRC Institute of Molecular Oncology Foundation, IFOM-IEO Campus, Via Adamello 16, Milano 20139, Italy 2 Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen 45122, Germany; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-2-574303306; Fax: +39-2-574303231. Academic Editor: Peter Frank Received: 31 March 2015 / Accepted: 18 June 2015 / Published: 23 June 2015 Abstract: Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles.
    [Show full text]
  • Suppression of Non-Homologous End Joining Does Not Rescue DNA Repair Defects
    bioRxiv preprint doi: https://doi.org/10.1101/151472; this version posted June 17, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Suppression of non-homologous end joining does not rescue DNA repair defects in Fanconi anemia patient cells Supawat Thongthip*, Brooke A. Conti*, Francis P. Lach and Agata Smogorzewska# Laboratory of Genome Maintenance, The Rockefeller University, New York, New York 10065, USA *These authors contributed equally to this paper #Correspondence: [email protected] Keywords: Fanconi anemia, non-homologous end joining, FANCA, DNA-PKcs, LIGIV, KU70, KU80, 53BP1, mitomycin C. 1 bioRxiv preprint doi: https://doi.org/10.1101/151472; this version posted June 17, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT Severe cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clusterd Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.
    [Show full text]
  • Cell Cycle- and DNA Repair Pathway-Specific Effects of Apoptosis on Tumor Suppression
    Cell cycle- and DNA repair pathway-specific effects of apoptosis on tumor suppression Steven S. Fostera, Saurav Dea, Linda K. Johnsonb, John H. J. Petrinia,1, and Travis H. Strackera,c,1 aMolecular Biology Program, Sloan-Kettering Institute, New York, NY 10021; bCenter of Comparative Medicine and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and cOncology Programme, Institute for Research in Biomedicine, 08028 Barcelona, Spain Edited by James E. Haber, Brandeis University, Waltham, MA, and approved May 8, 2012 (received for review January 12, 2012) The DNA damage response comprises DNA repair, cell-cycle check- required for subsequent activation of ATM, an apical kinase in point control, and DNA damage-induced apoptosis that collectively the DDR (2). ATM and related kinases are estimated to modify promote genomic integrity and suppress tumorigenesis. Previ- more than 700 substrates in response to DNA damage on con- ously, we have shown that the Chk2 kinase functions indepen- sensus SQ/TQ sites (6, 7). Among these substrates are key regu- dently of the Mre11 complex (Mre11, Rad50, and Nbs1) and ATM lators of cell-cycle checkpoints, DNA repair factors, and proteins in apoptosis and suppresses tumorigenesis resulting from hypo- that regulate apoptosis and senescence, accounting for the broad morphic alleles of Mre11 or Nbs1. Based on this work, we have physiological impact of DDR functions (8). proposed that Chk2 limits the oncogenic potential of replication- Arguably one of the most important ATM substrates is the associated DNA damage. Here we further address the role of Chk2 tumor suppressor p53. p53 controls the G1/S cell-cycle check- and damage-induced apoptosis in suppressing the oncogenic po- point and is required for both apoptosis and senescence in various cellular contexts (9).
    [Show full text]
  • End-Joining Repair of Double-Strand Breaks in Drosophila Melanogaster Is Largely DNA Ligase IV Independent
    Copyright 2004 by the Genetics Society of America DOI: 10.1534/genetics.104.033902 End-Joining Repair of Double-Strand Breaks in Drosophila melanogaster Is Largely DNA Ligase IV Independent Mitch McVey,*,†,1 Dora Radut* and Jeff J. Sekelsky*,‡ *Department of Biology, †SPIRE Program and ‡Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 27599 Manuscript received July 23, 2004 Accepted for publication August 26, 2004 ABSTRACT Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4- independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies.
    [Show full text]