Radiation-Induced Chromosome Aberrations in Saccharomyces cerevisiae: Influence of DNA Repair Pathways

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 975-988 ◽  
Author(s):  
Anna A Friedl ◽  
Markus Kiechle ◽  
Barbara Fellerhoff ◽  
Friederike Eckardt-Schupp
Keyword(s):  
Homologous Recombination ◽  
Chromosome Aberrations ◽  
Nonhomologous End Joining ◽  
Yeast Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Cell Clones ◽  
Radiation Induced ◽  
The Relationship

AbstractRadiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(=YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.

scholarly journals Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology

2007 ◽  
Vol 6 (10) ◽  
pp. 1773-1781 ◽  
Author(s):  
Peter Burton ◽  
David J. McBride ◽  
Jonathan M. Wilkes ◽  
J. David Barry ◽  
Richard McCulloch
Keyword(s):  
Homologous Recombination ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Bacterial Species ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Ligase Iv

ABSTRACT DNA double-strand breaks (DSBs) are repaired primarily by two distinct pathways: homologous recombination and nonhomologous end joining (NHEJ). NHEJ has been found in all eukaryotes examined to date and has been described recently for some bacterial species, illustrating its ancestry. Trypanosoma brucei is a divergent eukaryotic protist that evades host immunity by antigenic variation, a process in which homologous recombination plays a crucial function. While homologous recombination has been examined in some detail in T. brucei, little work has been done to examine what other DSB repair pathways the parasite utilizes. Here we show that T. brucei cell extracts support the end joining of linear DNA molecules. These reactions are independent of the Ku heterodimer, indicating that they are distinct from NHEJ, and are guided by sequence microhomology. We also demonstrate bioinformatically that T. brucei, in common with other kinetoplastids, does not encode recognizable homologues of DNA ligase IV or XRCC4, suggesting that NHEJ is either absent or mechanistically diverged in these pathogens.

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

scholarly journals Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

2010 ◽  
Vol 30 (13) ◽  
pp. 3371-3383 ◽  
Author(s):  
Atsushi Shibata ◽  
Olivia Barton ◽  
Angela T. Noon ◽  
Kirsten Dahm ◽  
Dorothee Deckbar ◽  
...  
Keyword(s):  
Chromosome Breakage ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Radiation Induced ◽  
High Doses ◽  
Interfering Rna ◽  
Atm Signaling

ABSTRACT ATM-dependent initiation of the radiation-induced G2/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G2 phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G2/M checkpoint is maintained in irradiated G2 cells, in light of our current understanding of G2 phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1 −/− and MDC1 −/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

scholarly journals Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells

2002 ◽  
Vol 22 (16) ◽  
pp. 5869-5878 ◽  
Author(s):  
Cecilia Lundin ◽  
Klaus Erixon ◽  
Catherine Arnaudeau ◽  
Niklas Schultz ◽  
Dag Jenssen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Lethal Effects ◽  
Strand Breaks ◽  
Replication Arrest

ABSTRACT Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

scholarly journals A game of musical chairs: Pro- and anti-resection factors compete for TOPBP1 binding after DNA damage

2017 ◽  
Vol 216 (3) ◽  
pp. 535-537 ◽  
Author(s):  
Kenji Shimada ◽  
Susan M. Gasser
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Binding Sites ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Pathway Regulation

DNA double strand breaks (DSBs) are generally repaired through nonhomologous end joining or homologous recombination. In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607031) report that the conserved scaffold protein TOPBP1Dpb11 provides binding sites for both pro- and anti-resection factors at DSBs, providing insights into repair pathway regulation.

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.

scholarly journals DNA Damage-Inducible and RAD52-Independent Repair of DNA Double-Strand Breaks in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1085-1099 ◽  
Author(s):  
Carol Wood Moore ◽  
Judith McKoy ◽  
Michelle Dardalhon ◽  
Darline Davermann ◽  
Marcia Martinez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Strand Breaks ◽  
Mutant Strains

Abstract Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 γ irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following γ irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by γ irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after γ irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

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