Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast.

Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over.

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Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus.

Genetics ◽

10.1093/genetics/130.3.451 ◽

1992 ◽

Vol 130 (3) ◽

pp. 451-460 ◽

Cited By ~ 10

Author(s):

A Plessis ◽

A Perrin ◽

J E Haber ◽

B Dujon

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Lacz Gene ◽

Direct Repeats ◽

Strand Breaks ◽

Group I ◽

Break Formation ◽

Target Plasmid ◽

Gene Conversions

Abstract The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega+ intron to omega- DNA. We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate. We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination. With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions. Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene. We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated. Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps.

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CXXC1 is redundant for normal DNA double-strand break formation and meiotic recombination in mouse

10.1101/305508 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Tian ◽

Timothy Billings ◽

Petko M. Petkov

Keyword(s):

Dna Sequences ◽

Meiotic Recombination ◽

Strand Break ◽

Double Strand Break ◽

Conditional Knockout ◽

Strand Breaks ◽

Conditional Knockout Mouse ◽

Dna Double Strand Break ◽

Chromosome Axis ◽

Break Formation

AbstractIn most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break initiation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here we investigated whether CXXC1 has a similar function in mouse meiosis. We found that CXXC1 is co-expressed and interacts with PRDM9 in mouse spermatocytes. To investigate the meiotic function of CXXC1, we created a Cxxc1 conditional knockout mouse to deplete CXXC1 before the onset of meiosis. Surprisingly, knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation activity, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.Author SummaryMeiotic recombination increases genetic diversity by ensuring novel combination of alleles passing onto the next generation correctly. In most mammals, the meiotic recombination sites are determined by histone methyltransferase PRDM9. These sites subsequently become associated with the chromosome axis with the participation of additional proteins and undergo double strand breaks, which are repaired by homologous recombination. In Saccharomyces cerevisiae, Spp1 (ortholog of CXXC1) binds to methylated H3K4 and connects these sites with chromosome axis promoting DSB formation. However, our data suggest that even though CXXC1 interacts with PRDM9 in male germ cells, it does not play a crucial role in mouse meiotic recombination. These results indicate that, unlike in S. cerevisiae, a recombination initiation pathway that includes CXXC1 could only serve as a non-essential pathway in mouse meiotic recombination.

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Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

Nucleic Acids Research ◽

10.1093/nar/gkaa597 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8490-8508 ◽

Cited By ~ 1

Author(s):

Sarah S Henrikus ◽

Camille Henry ◽

Amy E McGrath ◽

Slobodan Jergic ◽

John P McDonald ◽

...

Keyword(s):

Dna Polymerase ◽

Single Molecule ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Strand Breaks ◽

E Coli ◽

Pol Iv ◽

Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

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A global view of meiotic double-strand break end resection

Science ◽

10.1126/science.aak9704 ◽

2017 ◽

Vol 355 (6320) ◽

pp. 40-45 ◽

Cited By ~ 79

Author(s):

Eleni P. Mimitou ◽

Shintaro Yamada ◽

Scott Keeney

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Naked Dna ◽

Global View ◽

Genome Wide ◽

End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

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An Immortalised Leukaemia Cell Line Models the Quiescence of Leukaemic Stem Cells and Shows Impaired Double Strand Break Repair Following Daunorubicin Treatment

Blood ◽

10.1182/blood.v116.21.3989.3989 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3989-3989

Author(s):

Claire Seedhouse ◽

Abigail Whittall ◽

Karuna Tandon ◽

Nigel H. Russell ◽

Monica Pallis

Keyword(s):

Cell Cycle ◽

Strand Break ◽

Multidrug Resistant ◽

Double Strand Break ◽

Double Strand Breaks ◽

Proliferating Cells ◽

Drug Induced ◽

Strand Breaks ◽

Damage Response ◽

Mitochondrial Pore

Abstract Abstract 3989 Objective: Approximately 50% of patients with acute myeloid leukaemia respond to remission-induction chemotherapy, but later relapse. Relapse is thought to be due to the continued presence of a quiescent, chemoresistant leukaemic cell subpopulation. Understanding the damage response in these cells might help to guide targeted therapies. We therefore developed an in vitro model of the quiescent subpopulation and used it to study drug-induced damage and repair in quiescent multidrug resistant cells. Methods: We cultured CD34+ CD38- multidrug resistant KG1a AML cells under several conditions reported to induce cell cycle arrest. We used Pyronin Y to measure RNA content and 7-aminoactinomycin D to measure cell viability. Chemosensitivity, reactive oxygen species (ROS), mitochondrial pore transition and oxidative damage were measured flow cytometrically. gammaH2A.X foci were quantified to measure the double strand break response and DNA damage response and repair gene expression was studied using PCR microarrays and confirmed by real time PCR. Results: mTOR inhibitors induced an increase in G0 without induction of apoptosis. 48 hours' exposure to rapamycin increased the proportion of G0 cells from 13.3% (SD 2.3%) to 46.1% (SD 6%) and decreased mean cell volume. Delayed re-entry into cell cycle following rapamycin withdrawal confirmed the G0 status of these cells. Differentiation markers remained negative. Although several of the other conditions studied resulted in reduced cell growth, they also induced apoptosis, as did combinations of rapamycin with other growth inhibitors. The toxicity of the chemotherapy drug daunorubicin, which acts in part by inducing ROS, was reduced in the quiescence-enriched cells. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that apoptotic pathways are not impaired. However, both basal and drug-induced ROS were significantly lower in quiescence-enriched than in the proliferating cells (p=0.006 for basal ROS and 0.013 for daunorubicin-induced ROS). Furthermore, several DNA repair genes were differentially regulated following daunorubicin treatment of the quiescence-enriched compared to the proliferating cells – these included genes responsible for the repair of double strand breaks. On treatment with daunorubicin, double strand breaks, but not oxidative damage to DNA were observed in both cell populations. However, strikingly, although quiescence-enriched cells sustained fewer DNA damage foci than proliferating cells, they were unable to resolve the damage after daunorubicin was removed. Conclusion: By using rapamycin to enrich KG1a cells for quiescence, we have shown low basal and drug-induced ROS to be associated with chemoresistance in these cells. However, we also found that quiescence gave rise to an impaired double strand break response, which might force these cells to rely on alternative repair pathways and thus be sensitive to synthetic lethal targeting. Disclosures: No relevant conflicts of interest to declare.

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Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

Journal of Bacteriology ◽

10.1128/jb.00768-15 ◽

2015 ◽

Vol 198 (3) ◽

pp. 591-599 ◽

Cited By ~ 1

Author(s):

Fares Osam Yáñez-Cuna ◽

Mildred Castellanos ◽

David Romero

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Content Type ◽

Strand Breaks ◽

Biased Gene Conversion ◽

Resident Plasmid

ABSTRACTGene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family ofRhizobium etliwas analyzed by using a two-plasmid system, where each plasmid carried a copy ofnifH. One of thenifHcopies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced intoR. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that thenifHgene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into thenifHhomolog that had the RFLPs used for scoring gene conversion.In vivoexpression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into anR. etlistrain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs.IMPORTANCEIn this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of resident DNA and can be reversed upon introduction of a double-strand break on the incoming sequence. Since conditions used in this work are similar to those in horizontal gene transfer, it provides evidence that, upon transfer, the resident DNA preferentially acquires gene variants.

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SIRT6 is a DNA Double-Strand Break Sensor

10.1101/765172 ◽

2019 ◽

Author(s):

Lior Onn ◽

Miguel Portillo ◽

Stefan Ilic ◽

Gal Cleitman ◽

Daniel Stein ◽

...

Keyword(s):

Dna Damage ◽

Binding Capacity ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

The Road ◽

Non Homologous End Joining

AbstractDNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

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swi6, a gene required for mating-type switching, prohibits meiotic recombination in the mat2-mat3 "cold spot" of fission yeast.

Genetics ◽

10.1093/genetics/129.4.1033 ◽

1991 ◽

Vol 129 (4) ◽

pp. 1033-1042

Author(s):

A J Klar ◽

M J Bonaduce

Keyword(s):

Fission Yeast ◽

Mating Type ◽

Meiotic Recombination ◽

Hot Spot ◽

Strand Break ◽

Double Strand Break ◽

Cold Spot ◽

Additional Result ◽

Type Locus ◽

Mating Type Switching

Abstract Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.

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Control of meiotic double-strand-break formation by ATM: local and global views

10.1101/236984 ◽

2017 ◽

Cited By ~ 1

Author(s):

Agnieszka Lukaszewicz ◽

Julian Lange ◽

Scott Keeney ◽

Maria Jasin

Keyword(s):

Strand Break ◽

Double Strand Breaks ◽

Genome Integrity ◽

Future Research ◽

Dna Double Strand Breaks ◽

Time Loss ◽

Strand Breaks ◽

Research Perspectives ◽

The Right ◽

Break Formation

ABSTRACTDNA double-strand breaks (DSBs) generated by the SPO11 protein initiate meiotic recombination, an essential process for successful chromosome segregation during gametogenesis. The activity of SPO11 is controlled by multiple factors and regulatory mechanisms, such that the number of DSBs is limited and DSBs form at distinct positions in the genome and at the right time. Loss of this control can affect genome integrity or cause meiotic arrest by mechanisms that are not fully understood. Here we focus on the DSB-responsive kinase ATM and its functions in regulating meiotic DSB numbers and distribution. We review the recently discovered roles of ATM in this context, discuss their evolutionary conservation, and examine future research perspectives.

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