scholarly journals The Multifaceted Roles of Ku70/80

2021 ◽  
Vol 22 (8) ◽  
pp. 4134
Author(s):  
Sayma Zahid ◽  
Murielle Seif El Dahan ◽  
Florence Iehl ◽  
Paloma Fernandez-Varela ◽  
Marie-Helene Le Du ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Nuclease Activity ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna And Rna ◽  
Central Actor ◽  
Dna Ends

DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.

scholarly journals Loss of autophagy causes a synthetic lethal deficiency in DNA repair

2015 ◽  
Vol 112 (3) ◽  
pp. 773-778 ◽  
Author(s):  
Emma Y. Liu ◽  
Naihan Xu ◽  
Jim O’Prey ◽  
Laurence Y. Lao ◽  
Sanket Joshi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Repair Process ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Cytoplasmic Process ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Genomic Damage

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Benjamin M. Stinson ◽  
Joseph J. Loparo
Keyword(s):  
Genome Stability ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Annual Review ◽  
Publication Date ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Break Site ◽  
Dna Ends

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

2015 ◽  
Vol 35 (14) ◽  
pp. 2448-2463 ◽  
Author(s):  
Matthew Jessulat ◽  
Ramy H. Malty ◽  
Diem-Hang Nguyen-Tran ◽  
Viktor Deineko ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Large Scale ◽  
Spindle Assembly Checkpoint ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Cell Cycle Regulators ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Content Type

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.

scholarly journals Role of the Nuclease Activity ofSaccharomyces cerevisiaeMre11 in Repair of DNA Double-Strand Breaks in Mitotic Cells

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1701-1713 ◽  
Author(s):  
L Kevin Lewis ◽  
Francesca Storici ◽  
Stephen Van Komen ◽  
Shanna Calero ◽  
Patrick Sung ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Complex Functions ◽  
Mitotic Cells

AbstractThe Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.

DNA End Resection: Mechanism and Control

2021 ◽  
Vol 55 (1) ◽  
pp. 285-307
Author(s):  
Petr Cejka ◽  
Lorraine S. Symington
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Critical Determinant ◽  
Strand Breaks ◽  
Dna End Resection ◽  
And Control ◽  
End Resection ◽  
Dna Ends

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5′→3′ nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

scholarly journals Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Noriko Ishida ◽  
Tadashi Nakagawa ◽  
Shun-Ichiro Iemura ◽  
Akira Yasui ◽  
Hiroki Shima ◽  
...  
Keyword(s):  
Dna Repair ◽  
Ubiquitin Ligase ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Arginine Residues ◽  
Prolonged Retention ◽  
Damaged Dna

ABSTRACT Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.

scholarly journals ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9710-9723
Author(s):  
Sébastien Britton ◽  
Pauline Chanut ◽  
Christine Delteil ◽  
Nadia Barboule ◽  
Philippe Frit ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Dna Strand ◽  
Dna Ends

Abstract Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

scholarly journals XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

2015 ◽  
Vol 35 (17) ◽  
pp. 3017-3028 ◽  
Author(s):  
Sunetra Roy ◽  
Abinadabe J. de Melo ◽  
Yao Xu ◽  
Satish K. Tadi ◽  
Aurélie Négrel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Cell Strains ◽  
Dna Ends

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA endsin vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNAin vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

scholarly journals Differential Suppression of DNA Repair Deficiencies of Yeast rad50, mre11 and xrs2 Mutants by EXO1 and TLC1 (the RNA Component of Telomerase)

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 49-62 ◽  
Author(s):  
L Kevin Lewis ◽  
G Karthikeyan ◽  
James W Westmoreland ◽  
Michael A Resnick
Keyword(s):  
Dna Repair ◽  
Nuclease Activity ◽  
Nonhomologous End Joining ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mutant Proteins ◽  
Repair Deficiency ◽  
Yeast Telomerase ◽  
Dna Repair Deficiency

Abstract Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5′–3′ exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.

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