The Role of Nonhomologous DNA End Joining, Conservative Homologous Recombination, and Single-Strand Annealing in the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by H2O2in Mammalian Cells

2008 ◽  
Vol 170 (6) ◽  
pp. 784-793 ◽  
Author(s):  
Marlis Frankenberg-Schwager ◽  
Manuela Becker ◽  
Irmgard Garg ◽  
Elke Pralle ◽  
Hartmut Wolf ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Cycle Dependent

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 Non-homologous DNA end joining.

2003 ◽  
Vol 50 (4) ◽  
pp. 891-908 ◽  
Author(s):  
Elzbieta Pastwa ◽  
Janusz Błasiak
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Repetitive Sequences ◽  
Protein A ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Sir Proteins ◽  
Single Strand Annealing ◽  
Ku Protein ◽  
Strand Annealing

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.

scholarly journals Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1671 ◽  
Author(s):  
George Iliakis ◽  
Emil Mladenov ◽  
Veronika Mladenova
Keyword(s):  
High Speed ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Single Strand Annealing ◽  
Modes Of Processing ◽  
Non Homologous End Joining ◽  
Strand Annealing

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.

scholarly journals Multiple Pathways for Repair of DNA Double-Strand Breaks in Mammalian Chromosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8353-8360 ◽  
Author(s):  
Yunfu Lin ◽  
Tamas Lukacsovich ◽  
Alan S. Waldman
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Base Pairs ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks

2005 ◽  
Vol 8 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Ali Jazayeri ◽  
Jacob Falck ◽  
Claudia Lukas ◽  
Jiri Bartek ◽  
Graeme C. M. Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Dependent

scholarly journals Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

2016 ◽  
Vol 113 (16) ◽  
pp. 4308-4313 ◽  
Author(s):  
Seiji N. Sugiman-Marangos ◽  
Yoni M. Weiss ◽  
Murray S. Junop
Keyword(s):  
Deinococcus Radiodurans ◽  
Double Strand Breaks ◽  
Single Strand ◽  
High Fidelity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Single Strand Annealing ◽  
Dna Strands ◽  
Strand Annealing

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

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