scholarly journals Telomeric Double Strand Breaks Facilitate Formation of 5’ C-Rich Overhangs in G1 Human Cells

2019 ◽  
Author(s):  
Christopher B Nelson ◽  
Taghreed M Al Turki ◽  
Lynn Taylor ◽  
David G Maranon ◽  
Keiko Muraki ◽  
...  
Keyword(s):  
Cellular Response ◽  
Human Cells ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Telomeric Dna ◽  
Strand Breaks ◽  
Alternative Lengthening ◽  
Damage Response ◽  
Important Regulator

AbstractTelomeres are repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating an inappropriate DNA damage response (DDR). Here, we characterized the human cellular response to targeted telomeric DSBs in telomerase positive and telomerase-independent alternative lengthening of telomeres (ALT) cells, specifically in G1. Telomeric DSBs in G1 human cells elicited early signatures of a DDR, however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical nonhomologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensively resected tracks of 5’ C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells also resulted in significant increases in 5’ C-rich (ss)telomeric DNA in G1, which rather than RPA, were bound by the complementary telomeric RNA, TERRA. These results suggest that targeting TERRA-mediated protection at damaged telomeres may represent a promising therapeutic strategy, particularly against ALT-positive cancers.

scholarly journals Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5′ C-Rich Overhangs and Recruitment of TERRA

2021 ◽  
Vol 12 ◽  
Author(s):  
Christopher B. Nelson ◽  
Taghreed M. Alturki ◽  
Jared J. Luxton ◽  
Lynn E. Taylor ◽  
David G. Maranon ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Human Cells ◽  
Double Strand Breaks ◽  
End Joining ◽  
Telomeric Dna ◽  
Strand Breaks ◽  
Alternative Lengthening ◽  
Non Homologous End Joining ◽  
G1 Cells

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5′ C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5′ C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

scholarly journals No Overt Nucleosome Eviction at Deprotected Telomeres

2008 ◽  
Vol 28 (18) ◽  
pp. 5724-5735 ◽  
Author(s):  
Peng Wu ◽  
Titia de Lange
Keyword(s):  
Cellular Response ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Damage Response ◽  
Atr Kinase ◽  
Atm Signaling

ABSTRACT Dysfunctional telomeres elicit the canonical DNA damage response, which includes the activation of the ATM or ATR kinase signaling pathways and end processing by nonhomologous end joining (NHEJ) or homologous recombination (HR). The cellular response to DNA double-strand breaks has been proposed to involve chromatin remodeling and nucleosome eviction, but whether dysfunctional telomeres undergo chromatin reorganization is not known. Here, we report on the nucleosomal organization of telomeres that have become deprotected through the deletion of the shelterin components TRF2 or POT1. We found no evidence of changes in the nucleosomal organization of the telomeric chromatin or nucleosome eviction near the telomere terminus. An unaltered chromatin structure was observed at telomeres lacking TRF2, which activate the ATM kinase and are a substrate for NHEJ. Similarly, telomeres lacking POT1a and POT1b, which activate the ATR kinase, showed no overt nucleosome eviction. Finally, telomeres lacking TRF2 and Ku70, which are processed by HR, appeared to maintain their original nucleosomal organization. We conclude that ATM signaling, ATR signaling, NHEJ, and HR at deprotected telomeres can take place in the absence of overt nucleosome eviction.

scholarly journals Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

2005 ◽  
Vol 25 (3) ◽  
pp. 896-906 ◽  
Author(s):  
James M. Daley ◽  
Thomas E. Wilson
Keyword(s):  
Gc Content ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Primary Determinant ◽  
Overhang Length ◽  
Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

scholarly journals Regulation of DNA Double-Strand Break Repair by Non-Coding RNAs

2018 ◽  
Author(s):  
Roopa Thapar
Keyword(s):  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Protein Dna Interactions ◽  
Damage Response ◽  
Non Coding Rnas ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer.  Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs.  Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated.  This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair

scholarly journals The ATM protein: The importance of being active

2012 ◽  
Vol 198 (3) ◽  
pp. 273-275 ◽  
Author(s):  
Yosef Shiloh ◽  
Yael Ziv
Keyword(s):  
Cellular Response ◽  
Cancer Therapeutics ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Strand Breaks ◽  
Cell Biol ◽  
Response Network ◽  
Damage Response ◽  
Atm Protein

The ataxia telangiectasia mutated (ATM) protein kinase regulates the cellular response to deoxyribonucleic acid (DNA) double-strand breaks by phosphorylating numerous players in the extensive DNA damage response network. Two papers in this issue (Daniel et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204035; Yamamoto et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204098) strikingly show that, in mice, the presence of a catalytically inactive version of ATM is embryonically lethal. This is surprising because mice completely lacking ATM have a much more moderate phenotype. The findings impact on basic cancer research and cancer therapeutics.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

scholarly journals Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

2009 ◽  
Vol 184 (4) ◽  
pp. 515-526 ◽  
Author(s):  
Susan J. Hsiao ◽  
Susan Smith
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Adenosine Diphosphate ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
G2 Phase ◽  
Tankyrase 1

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

scholarly journals Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

2008 ◽  
Vol 205 (13) ◽  
pp. 3031-3040 ◽  
Author(s):  
Likun Du ◽  
Mirjam van der Burg ◽  
Sergey W. Popov ◽  
Ashwin Kotnis ◽  
Jacques J.M. van Dongen ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Igg Subclass ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

scholarly journals Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53

Oncogene ◽  
2005 ◽  
Vol 24 (10) ◽  
pp. 1663-1672 ◽  
Author(s):  
Jochen Dahm-Daphi ◽  
Petra Hubbe ◽  
Fruzsina Horvath ◽  
Raafat A El-Awady ◽  
Katie E Bouffard ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Site Specific ◽  
Strand Breaks ◽  
Radiation Induced ◽  
Wild Type P53

Pluronic copolymer encapsulated SCR7 as a potential anticancer agent

2015 ◽  
Vol 177 ◽  
pp. 155-161 ◽  
Author(s):  
Franklin John ◽  
Jinu George ◽  
Mrinal Srivastava ◽  
P. A. Hassan ◽  
V. K. Aswal ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Analytical Techniques ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Anticancer Properties

Nonhomologous end joining (NHEJ) of DNA double strand breaks (DSBs) inside cells can be selectively inhibited by 5,6-bis-(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) which possesses anticancer properties. The hydrophobicity of SCR7 decreases its bioavailability which is a major setback in the utilization of this compound as a therapeutic agent. In order to circumvent the drawback of SCR7, we prepared a polymer encapsulated form of SCR7. The physical interaction of SCR7 and Pluronic® copolymer is evident from different analytical techniques. The in vitro cytotoxicity of the drug formulations is established using the MTT assay.

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