scholarly journals The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Julianne Kitevski-LeBlanc ◽  
Amélie Fradet-Turcotte ◽  
Predrag Kukic ◽  
Marcus D Wilson ◽  
Guillem Portella ◽  
...  
Keyword(s):  
Interaction Mechanism ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
New Class ◽  
Break Site ◽  
Methyl Trosy ◽  
Resolution Model ◽  
Histone Ubiquitylation ◽  
Dynamics Simulations

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.

scholarly journals Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Alina Rakhimova ◽  
Seiji Ura ◽  
Duen-Wei Hsu ◽  
Hong-Yu Wang ◽  
Catherine J. Pears ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Histone H2b ◽  
Site Specific ◽  
Strand Breaks ◽  
Adp Ribosylation

scholarly journals Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea M. Kaminski ◽  
John M. Pryor ◽  
Dale A. Ramsden ◽  
Thomas A. Kunkel ◽  
Lars C. Pedersen ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Template Strand ◽  
Single Strand Breaks ◽  
Dna Double Strand Break ◽  
Break Site

Abstract Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

scholarly journals Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast

2020 ◽  
Vol 10 (10) ◽  
pp. 3821-3829
Author(s):  
Dionna Gamble ◽  
Samantha Shaltz ◽  
Sue Jinks-Robertson
Keyword(s):  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Strand Transfer ◽  
Repair Rate ◽  
Site Specific ◽  
Strand Breaks ◽  
Break Site ◽  
A Site ◽  
Enzyme I

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3′ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3′ end affects recombination outcomes and whether the presence of a 3′ vs. 5′ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3′ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3′ vs. 5′ overhangs differs, we compared the well-studied 3′ overhang created by I-SceI to a 5′ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

scholarly journals The tale of a tail: histone H4 acetylation and the repair of DNA breaks

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160284 ◽  
Author(s):  
Surbhi Dhar ◽  
Ozge Gursoy-Yuzugullu ◽  
Ramya Parasuram ◽  
Brendan D. Price
Keyword(s):  
Dna Repair ◽  
Open Chromatin ◽  
Histone H4 ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Histone H4 Acetylation ◽  
Break Site ◽  
Bromodomain Proteins ◽  
Complex Architecture

The ability of cells to detect and repair DNA double-strand breaks (DSBs) within the complex architecture of the genome requires co-ordination between the DNA repair machinery and chromatin remodelling complexes. This co-ordination is essential to process damaged chromatin and create open chromatin structures which are required for repair. Initially, there is a PARP-dependent recruitment of repressors, including HP1 and several H3K9 methyltransferases, and exchange of histone H2A.Z by the NuA4-Tip60 complex. This creates repressive chromatin at the DSB in which the tail of histone H4 is bound to the acidic patch on the nucleosome surface. These repressor complexes are then removed, allowing rapid acetylation of the H4 tail by Tip60. H4 acetylation blocks interaction between the H4 tail and the acidic patch on adjacent nucleosomes, decreasing inter-nucleosomal interactions and creating open chromatin. Further, the H4 tail is now free to recruit proteins such as 53BP1 to DSBs, a process modulated by H4 acetylation, and provides binding sites for bromodomain proteins, including ZMYND8 and BRD4, which are important for DSB repair. Here, we will discuss how the H4 tail functions as a dynamic hub that can be programmed through acetylation to alter chromatin packing and recruit repair proteins to the break site. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

Detecting, signalling and repairing DNA double-strand breaks

2001 ◽  
Vol 29 (6) ◽  
pp. 655-661 ◽  
Author(s):  
S. P. Jackson
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Double Strand Breaks ◽  
Model Organisms ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Site Specific ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Recombination Processes

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revelaed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

scholarly journals Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53

Oncogene ◽  
2005 ◽  
Vol 24 (10) ◽  
pp. 1663-1672 ◽  
Author(s):  
Jochen Dahm-Daphi ◽  
Petra Hubbe ◽  
Fruzsina Horvath ◽  
Raafat A El-Awady ◽  
Katie E Bouffard ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Site Specific ◽  
Strand Breaks ◽  
Radiation Induced ◽  
Wild Type P53

scholarly journals Recombinational repair of nuclease-generated mitotic double-strand breaks with different end structures in yeast

2020 ◽  
Author(s):  
Dionna Gamble ◽  
Samantha Shaltz ◽  
Sue Jinks-Robertson
Keyword(s):  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Strand Transfer ◽  
Repair Rate ◽  
Site Specific ◽  
Strand Breaks ◽  
Break Site ◽  
A Site ◽  
Enzyme I

ABSTRACTMitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3’ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3’ end affects recombination outcomes and whether the presence of a 3’ versus 5’ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers versus noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3’ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3’ versus 5’ overhangs differs, we compared the well-studied 3’ overhang created by I-SceI to a 5’ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN-than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

scholarly journals Homology-directed repair involves multiple strand invasion cycles in fission yeast

2020 ◽  
Author(s):  
Amanda J. Vines ◽  
Kenneth Cox ◽  
Bryan A. Leland ◽  
Megan C. King
Keyword(s):  
Fission Yeast ◽  
Homology Search ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Molecular Etiology ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Kinetics Of

AbstractHomology-directed repair of DNA double-strand breaks (DSBs) can be a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). While genetic studies highlight a key role for the RecQ helicase BLM/Rqh1 (in human and S. pombe cells, respectively) in promoting noncrossover repair, how homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, we find that the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair is likely to involve multiple site-specific and Rad51-dependent co-localization events between the DSB and donor sequence, suggesting that efficient interhomologue repair in fission yeast often involves multiple strand invasion events. By contrast, we find that loss of Rqh1 leads to successful repair through a single strand invasion event, suggesting that multiple strand invasion cycles reflect ongoing SDSA. However, failure to repair is also more likely in rqh1Δ cells, which could reflect increased strand invasion at non-homologous sites. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

scholarly journals BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair

2010 ◽  
Vol 191 (1) ◽  
pp. 45-60 ◽  
Author(s):  
Ismail Hassan Ismail ◽  
Christi Andrin ◽  
Darin McDonald ◽  
Michael J. Hendzel
Keyword(s):  
Radiation Sensitivity ◽  
Strand Break ◽  
Polycomb Group ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Additive Increase ◽  
Stem Cell Pluripotency ◽  
Histone Ubiquitylation ◽  
Polycomb Repressive Complex 1

Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability.

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