scholarly journals The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (11) ◽  
pp. 7681-7687 ◽  
Author(s):  
Debra A. Bressan ◽  
Bonnie K. Baxter ◽  
John H. J. Petrini
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Protein Complex ◽  
Cellular Response ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Synchronous Cultures ◽  
Dna Dsbs

ABSTRACT Saccharomyces cerevisiae mre11Δ mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Δ strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficientmre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.

A Double Strand Break during Telophase Is Repaired with Homologous Recombination Despite the Absence of an Available Sister Chromatid

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2421-2421
Author(s):  
Amit Patel ◽  
Luis Alcaide Aragon
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Conflicts Of Interest ◽  
Strand Break ◽  
Double Strand Break ◽  
Restriction Enzyme Digestion ◽  
Dsb Repair ◽  
Sister Chromatids ◽  
Kinetics Of ◽  
In Cells

Abstract Background: Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway. Methods: Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses. Results: Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase. The absence of an intact sister chromatid did not prevent DSB repair with the HR pathway during telophase. Nucleolytic resection was observed in the presence or absence of an intra-chromosomal homologous but non-identical DNA repair template. The DSB cut site did not become resistant to cycles of re-cleavage through loss of adjacent nucleotides. DSB repair by HR was dependent on Rad52. The kinetics of nucleolytic resection adjacent to the DSB, and repair by HR, were similar during telophase and metaphase. Conclusions: This is the first study to report the observation that the availability of the sister chromatid is not a requirement to promote DSB repair with the HR pathway during telophase. Initiation of HR occurs despite segregated sister chromatids, even in the absence of a non-identical homologous DNA donor template, with inherently mutagenic repair by HR. This unexpected discovery has important clinical implications to the pathogenesis of chromosomal translocations and oncogenesis, and tumour progression with repair of treatment-induced DSBs. Disclosures No relevant conflicts of interest to declare.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

scholarly journals DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

2018 ◽  
Author(s):  
Vijay R. Menon ◽  
Varsha Ananthapadmanabhan ◽  
Selene Swanson ◽  
Siddharth Saini ◽  
Fatmata Sesay ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Parp Inhibitor ◽  
Therapy Response ◽  
Strand Break ◽  
Dependent Manner ◽  
Dsb Repair ◽  
Altered Expression ◽  
Developmental Abnormalities

SummaryHumanDYRK1Agene encoding Dual-specificity tyrosine (Y)- Regulated Kinase 1A (DYRK1A) is a dosage-dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant of the DNA damaging therapy response.

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

scholarly journals Loss of BCL-3 sensitises colorectal cancer cells to DNA damage, revealing a role for BCL-3 in double strand break repair by homologous recombination

2021 ◽  
Author(s):  
Christopher Parker ◽  
Adam Christian Chambers ◽  
Dustin Flanagan ◽  
Tracey J Collard ◽  
Greg Ngo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Cellular Response ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Double Strand Break ◽  
Parp Inhibition ◽  
Adjuvant Chemoradiotherapy ◽  
Wild Type ◽  
Γ Irradiation

Objective: The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC) and increased expression of the gene correlates with poor patient prognosis. The aim is to investigate whether inhibiting BCL-3 can increase the response to DNA damage in CRC.Design: The function of BCL-3 in DNA damage response was studied in vitro using siRNA and CRISPR-Cas9 genome editing and in vivo using Bcl3-/- mice. DNA damage induced by γ-irradiation and/or cisplatin was quantified using H2AX and RAD51 foci, repair pathways investigated using HR/NHEJ assays and treatment with the PARP inhibitor olaparib. Result: Suppression of BCL-3 increases double strand break number and decreases homologous recombination in CRC cells, supported by reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/-mice, where the intestinal crypts of these mice exhibit sensitivity to DNA damage and a greater number of double strand breaks compared to wild type mice. FurthermoreApc.Kras-mutant x Bcl3-/- mice exhibit increased DNA damage and reduced RAD51+ cells compared to their wild type counterparts when treated with cisplatin. Conclusion: This work identifies BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjuvant to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.

scholarly journals The role of double-strand break-induced allelic homologous recombination in somatic plant cells

2002 ◽  
Vol 32 (3) ◽  
pp. 277-284 ◽  
Author(s):  
Brigitte Gisler ◽  
Siegfried Salomon ◽  
Holger Puchta
Keyword(s):  
Homologous Recombination ◽  
Plant Cells ◽  
Strand Break ◽  
Double Strand Break

scholarly journals Accurate Homologous Recombination Is a Prominent Double-Strand Break Repair Pathway in Mammalian Chromosomes and Is Modulated by Mismatch Repair Protein Msh2

2007 ◽  
Vol 27 (22) ◽  
pp. 7816-7827 ◽  
Author(s):  
Jason A. Smith ◽  
Laura A. Bannister ◽  
Vikram Bhattacharjee ◽  
Yibin Wang ◽  
Barbara Criscuolo Waldman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Lines ◽  
Fusion Gene ◽  
Sequence Divergence ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
Dsb Repair ◽  
Spontaneous Recombination ◽  
Mammalian Cell Lines

ABSTRACT We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.

scholarly journals The dual role of HOP2 in mammalian meiotic homologous recombination

2013 ◽  
Vol 42 (4) ◽  
pp. 2346-2357 ◽  
Author(s):  
Roberto J. Pezza ◽  
Oleg N. Voloshin ◽  
Alexander A. Volodin ◽  
Kingsley A. Boateng ◽  
Marina A. Bellani ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Homologous Chromosome ◽  
Strand Break ◽  
Dsb Repair ◽  
Chromosome Synapsis ◽  
Homologous Chromosome Pairing ◽  
Strand Invasion ◽  
High Level

Abstract Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.

scholarly journals Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange

2000 ◽  
Vol 20 (9) ◽  
pp. 3147-3156 ◽  
Author(s):  
Mies L. G. Dronkert ◽  
H. Berna Beverloo ◽  
Roger D. Johnson ◽  
Jan H. J. Hoeijmakers ◽  
Maria Jasin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Embryonic Stem ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54wild-type and knockout cells revealed direct evidence for a role ofmRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence ofmRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

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