Homolog-Dependent Repair Following Dicentric Chromosome Breakage in Drosophila melanogaster

Double-strand DNA breaks are repaired by one of several mechanisms that rejoin two broken ends. However, cells are challenged when asked to repair a single broken end and respond by: (1) inducing programmed cell death; (2) healing the broken end by constructing a new telomere; (3) adapting to the broken end and resuming the mitotic cycle without repair; and (4) using information from the sister chromatid or homologous chromosome to restore a normal chromosome terminus. During one form of homolog-dependent repair in yeast, termed break-induced replication (BIR), a template chromosome can be copied for hundreds of kilobases. BIR efficiency depends on Pif1 helicase and Pol32, a nonessential subunit of DNA polymerase δ. To date, there is little evidence that BIR can be used for extensive chromosome repair in higher eukaryotes. We report that a dicentric chromosome broken in mitosis in the male germline of Drosophila melanogaster is usually repaired by healing, but can also be repaired in a homolog-dependent fashion, restoring at least 1.3 Mb of terminal sequence information. This mode of repair is significantly reduced in pif1 and pol32 mutants. Formally, the repaired chromosomes are recombinants. However, the absence of reciprocal recombinants and the dependence on Pif1 and Pol32 strongly support the hypothesis that BIR is the mechanism for restoration of the chromosome terminus. In contrast to yeast, pif1 mutants in Drosophila exhibit a reduced rate of chromosome healing, likely owing to fundamental differences in telomeres between these organisms.

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53BP1 and BRCA1 control pathway choice for stalled replication restart

eLife ◽

10.7554/elife.30523 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Yixi Xu ◽

Shaokai Ning ◽

Zheng Wei ◽

Ran Xu ◽

Xinlin Xu ◽

...

Keyword(s):

Genome Stability ◽

Dna Breaks ◽

Dsb Repair ◽

Independent Manner ◽

Double Strand Dna Breaks ◽

Replication Restart ◽

Tumor Prevention ◽

Pathway Choice ◽

Break Induced Replication ◽

Stalled Replication Forks

The cellular pathways that restart stalled replication forks are essential for genome stability and tumor prevention. However, how many of these pathways exist in cells and how these pathways are selectively activated remain unclear. Here, we describe two major fork restart pathways, and demonstrate that their selection is governed by 53BP1 and BRCA1, which are known to control the pathway choice to repair double-strand DNA breaks (DSBs). Specifically, 53BP1 promotes a fork cleavage-free pathway, whereas BRCA1 facilitates a break-induced replication (BIR) pathway coupled with SLX-MUS complex-mediated fork cleavage. The defect in the first pathway, but not DSB repair, in a 53BP1 mutant is largely corrected by disrupting BRCA1, and vice versa. Moreover, PLK1 temporally regulates the switch of these two pathways through enhancing the assembly of the SLX-MUS complex. Our results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a DSB repair-independent manner.

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Inverted DNA Repeats Channel Repair of Distant Double-Strand Breaks into Chromatid Fusions and Chromosomal Rearrangements

Molecular and Cellular Biology ◽

10.1128/mcb.01740-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2601-2614 ◽

Cited By ~ 70

Author(s):

Kelly VanHulle ◽

Francene J. Lemoine ◽

Vidhya Narayanan ◽

Brandon Downing ◽

Krista Hull ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Inverted Repeats ◽

Dna Repeats ◽

Dna Breaks ◽

Strand Breaks ◽

Gross Chromosomal Rearrangements ◽

Double Strand Dna Breaks ◽

Single Strand Annealing ◽

Strand Annealing ◽

Break Induced Replication

ABSTRACT Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This “SSA-GCR” pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.

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Repair of DNA Breaks by Break-Induced Replication

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-081420-095551 ◽

2021 ◽

Vol 90 (1) ◽

Author(s):

Z.W. Kockler ◽

B. Osia ◽

R. Lee ◽

K. Musmaker ◽

A. Malkova

Keyword(s):

Genetic Material ◽

Annual Review ◽

Publication Date ◽

Dna Breaks ◽

Dsb Repair ◽

Double Strand Dna Breaks ◽

High Level ◽

Break Induced Replication ◽

Genome Destabilization

Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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The effect of sexual harassment on lethal mutation rate in female Drosophila melanogaster

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2012.1874 ◽

2013 ◽

Vol 280 (1750) ◽

pp. 20121874 ◽

Cited By ~ 14

Author(s):

Alexei A. Maklakov ◽

Simone Immler ◽

Hanne Løvlie ◽

Ilona Flis ◽

Urban Friberg

Keyword(s):

Dna Repair ◽

Drosophila Melanogaster ◽

Sexual Harassment ◽

Mutation Rate ◽

Population Level ◽

Dna Breaks ◽

Male Harassment ◽

Female Condition ◽

Double Strand Dna Breaks ◽

Repair Mechanisms

The rate by which new mutations are introduced into a population may have far-reaching implications for processes at the population level. Theory assumes that all individuals within a population have the same mutation rate, but this assumption may not be true. Compared with individuals in high condition, those in poor condition may have fewer resources available to invest in DNA repair, resulting in elevated mutation rates. Alternatively, environmentally induced stress can result in increased investment in DNA repair at the expense of reproduction. Here, we directly test whether sexual harassment by males, known to reduce female condition, affects female capacity to alleviate DNA damage in Drosophila melanogaster fruitflies. Female gametes can repair double-strand DNA breaks in sperm, which allows manipulating mutation rate independently from female condition. We show that male harassment strongly not only reduces female fecundity, but also reduces the yield of dominant lethal mutations, supporting the hypothesis that stressed organisms invest relatively more in repair mechanisms. We discuss our results in the light of previous research and suggest that social effects such as density and courtship can play an important and underappreciated role in mediating condition-dependent mutation rate.

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A chromosome breakage assay to monitor mitotic forces in budding yeast

Journal of Cell Science ◽

10.1242/jcs.107.4.891 ◽

1994 ◽

Vol 107 (4) ◽

pp. 891-902 ◽

Cited By ~ 1

Author(s):

J.A. Brock ◽

K. Bloom

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Budding Yeast ◽

Genetic Material ◽

Chromosome Breakage ◽

Dna Lesions ◽

Dicentric Chromosome ◽

Dna Breaks ◽

Dna Repair Gene ◽

Mitotic Delay

During the eukaryotic cell cycle, genetic material must be accurately duplicated and faithfully segregated to each daughter cell. Segregation of chromosomes is dependent on the centromere, a region of the chromosome which interacts with mitotic spindle microtubules during cell division. Centromere function in the budding yeast, Saccharomyces cerevisiae, can be regulated by placing an inducible promotor adjacent to centromere DNA. This conditional centromere can be integrated into chromosome III to generate a conditionally functional dicentric chromosome. Activation of the dicentric chromosome results in a transient mitotic delay followed by the generation of monocentric derivatives. The propagation of viable cells containing these monocentric derivative chromosomes is dependent upon the DNA repair gene RAD52, indicating that double-strand DNA breaks are structural intermediates in the dicentric repair pathway. We have used these conditionally dicentric chromosomes to monitor the exertion of mitotic forces during cell division. Analysis of synchronized cells reveal that lethality in dicentric, rad52 mutant cells occurs during G2/M phase and is concomitant with the transient mitotic delay. the delay is largely dependent upon the cell cycle checkpoint gene RAD9, which is involved in monitoring DNA damage. These data demonstrate that DNA lesions resulting from dicentric activation are responsible for signalling the mitotic delay. Since the delay precedes the decline of p34cdc28 kinase activity, mitotic forces sufficient to result in dicentric chromosome breakage are generated prior to spindle elongation and anaphase onset in yeast.

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Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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Faculty Opinions recommendation of Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717959211.793462621 ◽

2012 ◽

Author(s):

R Frank Kooy

Keyword(s):

Dna Breaks ◽

Genomic Architecture ◽

Break Induced Replication

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Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated With an Insertion of Telomeric Sequences Upstream of the HIS4 Locus in Yeast

Genetics ◽

10.1093/genetics/143.3.1115 ◽

1996 ◽

Vol 143 (3) ◽

pp. 1115-1125 ◽

Cited By ~ 3

Author(s):

Fei Xu ◽

Thomas D Petes

Keyword(s):

Meiotic Recombination ◽

Hydroxyl Groups ◽

Sequence Specificity ◽

Structure Mapping ◽

Dna Breaks ◽

Exonuclease Iii ◽

Recombination Hotspot ◽

Telomeric Sequences ◽

Double Strand Dna Breaks ◽

Double Strand Dna

Abstract Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3′-hydroxyl groups.

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Competition Between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/145.3.661 ◽

1997 ◽

Vol 145 (3) ◽

pp. 661-670 ◽

Cited By ~ 1

Author(s):

Qing-Qing Fan ◽

Fei Xu ◽

Michael A White ◽

Thomas D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Telomeric Sequence ◽

Coding Region ◽

Dna Breaks ◽

Local Formation ◽

Yeast Saccharomyces Cerevisiae ◽

Recombination Hotspots ◽

Double Strand Dna Breaks ◽

His4 Gene

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

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