Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Delisa E. Clay ◽  
Heidi S. Bretscher ◽  
Erin A. Jezuit ◽  
Korie B. Bush ◽  
Donald T. Fox
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Chromosome Segregation ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damage Signaling ◽  
Alternative End Joining

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

scholarly journals Persistent DNA Repair Signaling and DNA Polymerase Theta Promote Broken Chromosome Segregation

2021 ◽  
Author(s):  
Delisa E Clay ◽  
Heidi S Bretscher ◽  
Erin Jezuit ◽  
Korie Bush ◽  
Donald T Fox
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Polymerase ◽  
Chromosome Segregation ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Alternative End Joining ◽  
Segregation Of Chromosomes

Cycling cells must respond to double-strand breaks (DSBs) to avoid genome instability. Mis-segregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophila papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early-acting repair machinery (Mre11 and RPA3) to DSBs. This machinery persists as foci on DSBs as cells enter mitosis. Repair foci are resolved in a step-wise manner during mitosis. Repair signaling kinetics at DSBs depends on both monoubiquitination of the Fanconi Anemia (FA) protein Fancd2 and the alternative end- joining protein DNA Polymerase Theta. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

scholarly journals DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural Compounds

2021 ◽  
Author(s):  
Donna Goodenow ◽  
Kiran Lalwani ◽  
Christine Richardson
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Environmental Agents ◽  
Repair Mechanisms ◽  
Alternative End Joining ◽  
Differentiation Status

Eukaryotic cells use homologous recombination (HR), classical end-joining (C-NHEJ), and alternative end-joining (Alt-EJ) to repair DNA double-strand breaks (DSBs). Repair pathway choice is controlled by the activation and activity of pathways specific proteins in eukaryotes. Activity may be regulated by cell cycle stage, tissue type, and differentiation status. Bioflavonoids and other environmental agents such as pesticides have been shown to biochemically act as inhibitors of topoisomerase II (Top2). In cells, bioflavonoids directly lead to DNA double-strand breaks through both Top2-dependent and independent mechanisms, as well as induce DNA damage response (DDR) signaling, and promote alternative end-joining and chromosome alterations. This chapter will present differences in expression and activity of proteins in major DNA repair pathways, findings of Top2 inhibition by bioflavonoids and cellular response, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability and human disease.

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

DNA Repair ◽  
2015 ◽  
Vol 31 ◽  
pp. 29-40 ◽  
Author(s):  
Mario Moscariello ◽  
Radi Wieloch ◽  
Aya Kurosawa ◽  
Fanghua Li ◽  
Noritaka Adachi ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Alternative End Joining ◽  
Radiation Induced

scholarly journals Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

2015 ◽  
Vol 112 (24) ◽  
pp. 7507-7512 ◽  
Author(s):  
Ozge Gursoy-Yuzugullu ◽  
Marina K. Ayrapetov ◽  
Brendan D. Price
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Histone H4 ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Nucleosome Dynamics ◽  
Chromatin Template ◽  
End Resection

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

scholarly journals DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

2015 ◽  
Vol 112 (50) ◽  
pp. E6907-E6916 ◽  
Author(s):  
Damon Meyer ◽  
Becky Xu Hua Fu ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Dna Polymerase Delta ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Efficiency ◽  
Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

scholarly journals SIRT6 is a DNA Double-Strand Break Sensor

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.

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 Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

2006 ◽  
Vol 172 (6) ◽  
pp. 823-834 ◽  
Author(s):  
Michael J. Kruhlak ◽  
Arkady Celeste ◽  
Graham Dellaire ◽  
Oscar Fernandez-Capetillo ◽  
Waltraud G. Müller ◽  
...  
Keyword(s):  
Dna Damage ◽  
Living Cells ◽  
Double Strand Breaks ◽  
Dna Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Limited Mobility ◽  
Energy Filtering ◽  
Dna Damage Signaling ◽  
Cell Biol

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion (Pilch, D.R., O.A. Sedelnikova, C. Redon, A. Celeste, A. Nussenzweig, and W.M. Bonner. 2003. Biochem. Cell Biol. 81:123–129; Morrison, A.J., and X. Shen. 2005. Cell Cycle. 4:568–571; van Attikum, H., and S.M. Gasser. 2005. Nat. Rev. Mol. Cell. Biol. 6:757–765). The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated (ATM; Bakkenist, C.J., and M.B. Kastan. 2003. Nature. 421:499–506). However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30–40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate–dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

scholarly journals Altered DNA ligase activity in human disease

Mutagenesis ◽  
2019 ◽  
Vol 35 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Alan E Tomkinson ◽  
Tasmin Naila ◽  
Seema Khattri Bhandari
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Double Strand Breaks ◽  
Dna Ligase ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Ligases ◽  
Ligase Iv ◽  
Non Homologous End Joining

Abstract The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

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