scholarly journals Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

1998 ◽  
Vol 18 (4) ◽  
pp. 1891-1902 ◽  
Author(s):  
L. Kevin Lewis ◽  
Jakob M. Kirchner ◽  
Michael A. Resnick
Keyword(s):  
Nuclear Dna ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Physical And Chemical ◽  
Type Strains

ABSTRACT RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired inrad52 mutants and in G1-phase Rad+cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly,rad52 mutants were not more sensitive toEcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly whenEcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

DNA Repair ◽  
2007 ◽  
Vol 6 (5) ◽  
pp. 639-648 ◽  
Author(s):  
Yukitaka Katsura ◽  
Shigeru Sasaki ◽  
Masanori Sato ◽  
Kiyoshi Yamaoka ◽  
Kazumi Suzukawa ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

scholarly journals Repair of Endonuclease-Induced Double-Strand Breaks in Saccharomyces cerevisiae: Essential Role for Genes Associated with Nonhomologous End-Joining

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1513-1529 ◽  
Author(s):  
L Kevin Lewis ◽  
James W Westmoreland ◽  
Michael A Resnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Excision Repair ◽  
Cell Killing ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Chromosomal Dna ◽  
End Joining ◽  
Yeast Saccharomyces Cerevisiae ◽  
Strand Breaks

Abstract Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.

scholarly journals Frequency of DNA end joining in trans is not determined by the predamage spatial proximity of double-strand breaks in yeast

2019 ◽  
Vol 116 (19) ◽  
pp. 9481-9490 ◽  
Author(s):  
Sham Sunder ◽  
Thomas E. Wilson
Keyword(s):  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Spatial Distance ◽  
Spatial Proximity ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Efficiency ◽  
In Trans

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.

scholarly journals In Vivo Blunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining

2015 ◽  
Author(s):  
Jonathan M Geisinger ◽  
Sören Turan ◽  
Sophia Hernandez ◽  
Laura P Spector ◽  
Michele P Calos
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
Fluorescent Protein ◽  
Double Strand Breaks ◽  
Hek293 Cells ◽  
End Joining ◽  
Independent Manner ◽  
Strand Breaks ◽  
Non Homologous End Joining

The ability to precisely modify the genome in a site-specific manner is extremely useful. The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

scholarly journals Additive Effects of SbcCD and PolX Deficiencies in the In Vivo Repair of DNA Double-Strand Breaks in Deinococcus radiodurans

2007 ◽  
Vol 189 (13) ◽  
pp. 4784-4790 ◽  
Author(s):  
Esma Bentchikou ◽  
Pascale Servant ◽  
Geneviève Coste ◽  
Suzanne Sommer
Keyword(s):  
Double Mutant ◽  
Deinococcus Radiodurans ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Repair System ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Strand Breaks

ABSTRACT Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant ΔsbcCD ΔpolX Dr (where Dr indicates D. radiodurans) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.

scholarly journals The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks

2005 ◽  
Vol 25 (10) ◽  
pp. 3934-3944 ◽  
Author(s):  
Eun Yong Shim ◽  
Jia-Lin Ma ◽  
Ji-Hyun Oum ◽  
Yvonne Yanez ◽  
Sang Eun Lee
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Genotoxic Stress ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks

ABSTRACT Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Δ maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

scholarly journals Increased BUB1B/BUBR1 expression contributes to aberrant DNA repair activity leading to resistance to DNA-damaging agents

Oncogene ◽  
2021 ◽  
Author(s):  
Kazumasa Komura ◽  
Teruo Inamoto ◽  
Takuya Tsujino ◽  
Yusuke Matsui ◽  
Tsuyoshi Konuma ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Repair Activity ◽  
A Cell

AbstractThere has been accumulating evidence for the clinical benefit of chemoradiation therapy (CRT), whereas mechanisms in CRT-recurrent clones derived from the primary tumor are still elusive. Herein, we identified an aberrant BUB1B/BUBR1 expression in CRT-recurrent clones in bladder cancer (BC) by comprehensive proteomic analysis. CRT-recurrent BC cells exhibited a cell-cycle-independent upregulation of BUB1B/BUBR1 expression rendering an enhanced DNA repair activity in response to DNA double-strand breaks (DSBs). With DNA repair analyses employing the CRISPR/cas9 system, we revealed that cells with aberrant BUB1B/BUBR1 expression dominantly exploit mutagenic nonhomologous end joining (NHEJ). We further found that phosphorylated ATM interacts with BUB1B/BUBR1 after ionizing radiation (IR) treatment, and the resistance to DSBs by increased BUB1B/BUBR1 depends on the functional ATM. In vivo, tumor growth of CRT-resistant T24R cells was abrogated by ATM inhibition using AZD0156. A dataset analysis identified FOXM1 as a putative BUB1B/BUBR1-targeting transcription factor causing its increased expression. These data collectively suggest a redundant role of BUB1B/BUBR1 underlying mutagenic NHEJ in an ATM-dependent manner, aside from the canonical activity of BUB1B/BUBR1 on the G2/M checkpoint, and offer novel clues to overcome CRT resistance.

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