Ubiquitin recognition by UBZ and UMI domains for DNA damage response

Double-strand break (DSB) and interstrand crosslink (ICL) are serious damages in DNA. Responses to these DNA damages include ubiquitination of damaged chromatin and other substrates, which recruit protein complexes required for DNA repair. Therefore, many proteins involved in DNA damage response contain ubiquitin-binding modules. For instance, a ubiquitin ligase RNF168, which catalyzes K63-linked polyubiquitination of histone H2A, contains two types of ubiquitin binding motifs, MIU (motif interacting with ubiquitin) and UIM (UIM and MIU-related Ub-binding domain). FAAP20, which recruits Fanconi anemia proteins (crosslink-repair factors), contains a UBZ (ubiquitin-binding zinc finger) domain. To date, mechanisms for ubiquitin recognition by UMI and UBZ domains have remained unclear. In this study, we determined crystal structures of RNF168 UMI and FAAP20 UBZ in complex with ubiquitin at 1.9 Å resolutions, respectively. SPR analyses using UMI and UBZ mutants, which were designed to disrupt Ub binding, confirmed that the observed interactions between Ub and UMI or UBZ are critical for binding. Our structure and the accompanying in-vitro structure-based mutagenesis experiments reveal the structural basis of these important recognition events.

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Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

Journal of Biological Chemistry ◽

10.1074/jbc.c900032200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16066-16070 ◽

Cited By ~ 88

Author(s):

Navasona Krishnan ◽

Dae Gwin Jeong ◽

Suk-Kyeong Jung ◽

Seong Eon Ryu ◽

Andrew Xiao ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Mammalian Cells ◽

Protein Tyrosine Phosphatases ◽

Histone H2a ◽

Eyes Absent ◽

Damage Repair ◽

Damage Response

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

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Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

The Journal of Cell Biology ◽

10.1083/jcb.200902150 ◽

2009 ◽

Vol 186 (6) ◽

pp. 835-847 ◽

Cited By ~ 125

Author(s):

Jurgen A. Marteijn ◽

Simon Bekker-Jensen ◽

Niels Mailand ◽

Hannes Lans ◽

Petra Schwertman ◽

...

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Dna Damage Response ◽

Excision Repair ◽

Strand Break ◽

Epigenetic Mark ◽

Histone H2a ◽

Nucleotide Excision ◽

Damage Response ◽

Damaged Dna

Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.

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Structural basis for phosphorylation-dependent signaling in the DNA-damage response

Biochemistry and Cell Biology ◽

10.1139/o05-153 ◽

2005 ◽

Vol 83 (6) ◽

pp. 721-727 ◽

Cited By ~ 22

Author(s):

R Scott Williams ◽

Nina Bernstein ◽

Megan S Lee ◽

Melissa L Rakovszky ◽

Diana Cui ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Protein Interactions ◽

Tandem Repeats ◽

Strand Break ◽

Structural Basis ◽

Fha Domain ◽

Flexible Mode ◽

Polynucleotide Kinase ◽

Damage Response

The response of eukaryotic cells to DNA damage requires a multitude of protein–protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.Key words: BRCA1, BRCT, PNK, FHA, polynucleotide kinase, breast cancer, phosphopeptide-protein interactions, DNA damage response.

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The DNA damage response regulates the oocyte pool in mammals

10.1101/816488 ◽

2019 ◽

Author(s):

Ana Martínez-Marchal ◽

Maria Teresa Guillot ◽

Mònica Ferrer ◽

Anna Guixé ◽

Montserrat Garcia-Caldés ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Adult Life ◽

Strand Break ◽

Oocyte Number ◽

Genome Wide ◽

Germ Cell Cysts ◽

Damage Response

SummaryMammalian oogonia proliferate without completing cytokinesis producing germ cell cysts. Within these cysts, oocytes differentiate and enter meiosis, promote genome-wide double-strand break (DSBs) formation which repair by homologous recombination leads to synapsis of the homologous chromosomes. Errors in homologous recombination or synapsis trigger the activation of surveillance mechanisms, traditionally called ‘pachytene checkpoint’, to either repair them or send the cells to programmed death. Contrary to what is found in spermatocytes, most oocytes present a remarkable persistence of unrepaired DSBs at pachynema. Simultaneously, there is a massive oocyte death accompanying the oocyte cyst breakdown. This oocyte elimination is thought to be required to properly form the follicles, which constitute the pool of germ cells females will use during their adult life. Based on all the above mentioned, we hypothesized that the apparently inefficient meiotic recombination occurring in mouse oocytes may be required to eliminate most of the oocytes in order to regulate the oocyte number, promote cyst breakdown and follicle formation in mammalian females. To test this idea, we analyzed perinatal ovaries to evaluate the oocyte population, cyst breakdown and follicle formation in control and mutant mice for the effector kinase of the DNA damage response, CHK2. Our results confirm the involvement of CHK2 in the elimination of oocytes that accumulate unrepaired DSBs and show that CHK2 regulates the number of oocytes in fetal ovaries. We also show that CHK2 is required to eliminate oocytes as a result of LINE-1 activation, which was previously shown to be responsible for fetal oocyte loss. Nonetheless, the number of oocytes found in Chk2 mutant ovaries three days after birth was similar to that of control ovaries, suggesting the existence of CHK2-independent mechanisms capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2 mutant ovaries suggesting that CHK1 regulates postnatal oocyte death. Moreover, both CHK1 and CHK2 functions are required to timely breakdown cyst and form follicles. Altogether, we propose the DNA damage response controls the number of oocytes present perinatally and is required to properly break down oocyte cysts and form follicles, highlighting the importance of the DNA damage response in setting the reserve of oocytes each female will use during their entire lifespan.

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The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice

PLoS Genetics ◽

10.1371/journal.pgen.1009067 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009067

Author(s):

Ana Martínez-Marchal ◽

Yan Huang ◽

Maria Teresa Guillot-Ferriols ◽

Mònica Ferrer-Roda ◽

Anna Guixé ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Strand Break ◽

Double Strand Break ◽

Mutant Mice ◽

Oocyte Number ◽

Prophase I ◽

Damage Response

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

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Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

Journal of Experimental Medicine ◽

10.1084/jem.20131436 ◽

2014 ◽

Vol 211 (9) ◽

pp. 1759-1777 ◽

Cited By ~ 44

Author(s):

Cesare Lancini ◽

Paul C.M. van den Berk ◽

Joseph H.A. Vissers ◽

Gaetano Gargiulo ◽

Ji-Ying Song ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Genotoxic Stress ◽

Histone H2a ◽

Deubiquitinating Enzymes ◽

Dna Breaks ◽

Hematopoietic Stem ◽

Damage Response

Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3Δ/Δ HSCs. Beyond the hematopoietic system, Usp3Δ/Δ animals spontaneously developed tumors, and primary Usp3Δ/Δ cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress.

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The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

NAR Cancer ◽

10.1093/narcan/zcab002 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Luisa Statello ◽

Mohamad M Ali ◽

Silke Reischl ◽

Sagar Mahale ◽

Subazini Thankaswamy Kosalai ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cancer Biology ◽

Topoisomerase I ◽

Cell Cycle Checkpoints ◽

Topoisomerase 1 ◽

Damage Response ◽

Promote Cell Survival

Abstract Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1–cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

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PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2

Cell Death and Disease ◽

10.1038/s41419-020-2708-5 ◽

2020 ◽

Vol 11 (7) ◽

Author(s):

Nan Huang ◽

Chang Xu ◽

Liang Deng ◽

Xue Li ◽

Zhixuan Bian ◽

...

Keyword(s):

Dna Damage ◽

Histone Deacetylase ◽

Dna Damage Response ◽

De Novo ◽

Dna Damage Repair ◽

Histone Deacetylase 1 ◽

Damage Repair ◽

Damage Response

AbstractPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.

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Sites of Acetylation on Newly Synthesized Histone H4 Are Required for Chromatin Assembly and DNA Damage Response Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00460-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3286-3298 ◽

Cited By ~ 19

Author(s):

Zhongqi Ge ◽

Devi Nair ◽

Xiaoyan Guan ◽

Neha Rastogi ◽

Michael A. Freitas ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Histone H3 ◽

Model Organism ◽

Strand Break ◽

Chromatin Assembly ◽

Histone H4 ◽

Histone H4 Acetylation ◽

Damage Response ◽

A Site

The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.

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