scholarly journals Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

2020 ◽  
Vol 295 (45) ◽  
pp. 15378-15397
Author(s):  
Rujuta Yashodhan Gadgil ◽  
Eric J. Romer ◽  
Caitlin C. Goodman ◽  
S. Dean Rider ◽  
French J. Damewood ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Instability ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Kinase Gene ◽  
Strand Breaks ◽  
Restriction Cleavage ◽  
Break Induced Replication

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG)n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG)n chromosome fragility is repeat length–dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure–prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

scholarly journals Staphylococcal DNA repair is required for infection

2020 ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Human Blood ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Gram Positive ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Transposon Mutants

AbstractThe repair of DNA damage is essential for bacterial viability and contributes to adaptation via increased rates of mutation and recombination. However, the mechanisms by which DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks via the oxidative burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

scholarly journals Double-strand breaks are not the main cause of spontaneous sister chromatid exchange in wild-type yeast cells

2017 ◽  
Author(s):  
Clémence Claussin ◽  
David Porubský ◽  
Diana C.J. Spierings ◽  
Nancy Halsema ◽  
Stefan Rentas ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Genome Instability ◽  
Single Cells ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Template Strand ◽  
Sister Chromatids

SummaryHomologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. Contrary to what is commonly thought, we find that most spontaneous SCE events are not due to the repair of DNA double-strand breaks.

scholarly journals Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bente Benedict ◽  
Tanja van Harn ◽  
Marleen Dekker ◽  
Simone Hermsen ◽  
Asli Kucukosmanoglu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Origin Firing ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Cycle Arrest

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

scholarly journals Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

2019 ◽  
Vol 47 (6) ◽  
pp. 1609-1619 ◽  
Author(s):  
Qian Wu
Keyword(s):  
Single Molecule ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Structural Mechanism ◽  
Strand Breaks ◽  
Single Molecule Studies ◽  
Non Homologous End Joining ◽  
Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

scholarly journals FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Kasper Fugger ◽  
Wai Kit Chu ◽  
Peter Haahr ◽  
Arne Nedergaard Kousholt ◽  
Halfdan Beck ◽  
...  
Keyword(s):  
Cell Death ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals Current Understanding of RAD52 Functions: Fundamental and Therapeutic Insights

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 705
Author(s):  
Vanesa Gottifredi ◽  
Lisa Wiesmüller
Keyword(s):  
Homologous Recombination ◽  
Cancer Treatment ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Alternative Pathways ◽  
M Phase ◽  
Break Induced Replication

In this Special Issue, we would like to focus on the various functions of the RAD52 helicase-like protein and the current implications of such findings for cancer treatment. Over the last few years, various laboratories have discovered particular activities of mammalian RAD52—both in S and M phase—that are distinct from the auxiliary role of yeast RAD52 in homologous recombination. At DNA double-strand breaks, RAD52 was demonstrated to spur alternative pathways to compensate for the loss of homologous recombination functions. At collapsed replication forks, RAD52 activates break-induced replication. In the M phase, RAD52 promotes the finalization of DNA replication. Its compensatory role in the resolution of DNA double-strand breaks has put RAD52 in the focus of synthetic lethal strategies, which is particularly relevant for cancer treatment.

scholarly journals A role for nuclear envelope–bridging complexes in homology-directed repair

2014 ◽  
Vol 25 (16) ◽  
pp. 2461-2471 ◽  
Author(s):  
Rebecca K. Swartz ◽  
Elisa C. Rodriguez ◽  
Megan C. King
Keyword(s):  
Nuclear Envelope ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Linc Complex ◽  
Dna Double Strand Breaks ◽  
Cytoplasmic Microtubule ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Cytoplasmic Microtubules ◽  
Atr Kinase

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency.

scholarly journals DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160285 ◽  
Author(s):  
Magdalena B. Rother ◽  
Haico van Attikum
Keyword(s):  
Dna Repair ◽  
Posttranslational Modifications ◽  
Chromatin Structure ◽  
Hip Hop ◽  
Genome Stability ◽  
Genome Instability ◽  
Current Knowledge ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

scholarly journals Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

2019 ◽  
Author(s):  
Guillaume Gaullier ◽  
Genevieve Roberts ◽  
Uma M. Muthurajan ◽  
Samuel Bowerman ◽  
Johannes Rudolph ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Genome Instability ◽  
Structural Information ◽  
Regulatory Protein ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

AbstractPoly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

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