Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis

After exposure of mammalian cells to DNA damage, the endogenous Rad51 recombination protein is concentrated in multiple discrete foci, which are thought to represent nuclear domains for recombinational DNA repair. Overexpressed Rad51 protein forms foci and higher-order nuclear structures, even in the absence of DNA damage, in cells that do not undergo DNA replication synthesis. This correlates with increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21. Following DNA damage, constitutively Rad51-overexpressing cells show reduced numbers of DNA breaks and chromatid-type chromosome aberrations and a greater resistance to apoptosis. In contrast, Rad51 antisense inhibition reduces p21 protein levels and sensitizes cells to etoposide treatment. Downregulation of p21 inhibits Rad51 foci formation in both normal and Rad51-overexpressing cells. Collectively, our results show that Rad51 expression, Rad51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad51 protein and p21-mediated cell cycle regulation. This mechanism may contribute to a highly effective recombinational DNA repair in cell cycle-arrested cells and protection against DNA damage-induced apoptosis.

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ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1

Cell Death Discovery ◽

10.1038/s41420-021-00633-7 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Shuang Yan ◽

Man Song ◽

Jie Ping ◽

Shu-ting Lai ◽

Xiao-yu Cao ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Homologous Recombination ◽

Mammalian Cells ◽

Cell Cycle Checkpoint ◽

Dependent Manner ◽

Dna Breaks ◽

M Cell ◽

End Resection

AbstractTo maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

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Abundance of cyclin B1 regulates γ-radiation–induced apoptosis

Blood ◽

10.1182/blood.v95.8.2645 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2645-2650 ◽

Cited By ~ 114

Author(s):

Lisa A. Porter ◽

Gurmit Singh ◽

Jonathan M. Lee

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Mammalian Cells ◽

Hematopoietic Cells ◽

Cyclin B1 ◽

Regulatory Subunit ◽

Potent Inducer ◽

Γ Radiation ◽

Radiation Induced ◽

Induced Apoptosis

Abstract γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.

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Nonhomologous-End-Joining Factors Regulate DNA Repair Fidelity during Sleeping Beauty Element Transposition in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.23.23.8505-8518.2003 ◽

2003 ◽

Vol 23 (23) ◽

pp. 8505-8518 ◽

Cited By ~ 60

Author(s):

Stephen R. Yant ◽

Mark A. Kay

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Mammalian Cells ◽

Donor Site ◽

Protective Role ◽

Sleeping Beauty ◽

Nonhomologous End Joining ◽

Dna Breaks ◽

End Joining ◽

Transposon Insertion

ABSTRACT Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3′ overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

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Abundance of cyclin B1 regulates γ-radiation–induced apoptosis

Blood ◽

10.1182/blood.v95.8.2645.008k18_2645_2650 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2645-2650 ◽

Cited By ~ 5

Author(s):

Lisa A. Porter ◽

Gurmit Singh ◽

Jonathan M. Lee

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Mammalian Cells ◽

Hematopoietic Cells ◽

Cyclin B1 ◽

Regulatory Subunit ◽

Potent Inducer ◽

Γ Radiation ◽

Radiation Induced ◽

Induced Apoptosis

γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.

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Cell cycle repression and DNA repair defects follow constricted migration

10.1101/240838 ◽

2017 ◽

Author(s):

Charlotte R. Pfeifer ◽

Yuntao Xia ◽

Kuangzheng Zhu ◽

Dazhen Liu ◽

Jerome Irianto ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Nuclear Lamina ◽

Contact Inhibition ◽

Dna Breaks ◽

Lamin B1 ◽

Cell Cycle Dependence ◽

Repair Factor ◽

Cycle Dependence

AbstractCancer cell invasion into tissue or narrow capillaries often elongates the nucleus and sometimes damages it, but cell cycle effects are unknown and highly relevant to tumorigenesis. Here, nuclear rupture and DNA breaks caused by constricted migration are quantified in different phases of cell cycle - which is effectively repressed. Cancer lines with varying levels of contact inhibition and lamina proteins exhibit diverse frequencies of nuclear lamina rupture after migration, with prerupture dilation of gene-edited RFP-Lamin-B1 preceding DNA repair factor leakage in pressure-controlled distension. Post-migration rupture indeed associates with mis-localized DNA repair factors and increased DNA breaks as quantified by pan-nucleoplasmic foci of γH2AX, with foci counts always suppressed in late cell cycle. When contact-inhibited cells migrate through large pores into sparse microenvironments, cells re-enter cell cycle consistent with release from contact inhibition. In contrast, constricting pores effectively delay re-entry, but the excess DNA damage nonetheless exceeds any cell cycle dependence. Partial depletion of topoisomerase does not strongly affect cell cycle or the excess DNA damage, consistent with weak dependencies on replication stress. Constricted migration thus impacts cell cycle as well as DNA damage.

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Inhibition of Poly ADP Ribose Polymerase (PARP) Activity Exerts Cytotoxic Effects on Chromosomal Instability Syndrome and Leukaemic Cell Lines: Potential for Anti-Leukaemia Therapy.

Blood ◽

10.1182/blood.v108.11.2647.2647 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2647-2647

Author(s):

Terry J. Gaymes ◽

S. Shall ◽

Farzin Farzaneh ◽

Ghulam J. Mufti

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Cell Survival ◽

Cell Lines ◽

Chromosomal Instability ◽

Parp Inhibitors ◽

Dna Breaks ◽

Leukaemic Cells ◽

Parp Activity

Abstract Recent reports suggest that BrCA1−/− and BrCA2−/− cells can be selectively targeted for cell death through abrogation of their PARP activity. It is postulated that as a result of PARP inhibition, accumulation of single strand DNA breaks (SSB) leads to the replication fork collapse and conversion of SSB to double strand DNA breaks (DSB). The inability of repair defective cells such as BrCA2−/− to repair the DSB would lead to cell death. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a more specific and less toxic form of therapy for a number of haematological malignancies. Chromosomal instability (CI) syndromes that have inherent defects in double strand DNA repair also have a uniformly high incidence of transformation to acute leukaemia or lymphoma. In order to test the efficacy of PI therapy we analysed CI cell lines, myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) cell lines and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that cells from CI syndromes; Blooms syndrome, Fanconi Anaemia (FancD2 and FancA), Ataxia telancgectasia and Nijmegen break syndrome display abnormal cell cycle profiles and excessive apoptosis in response to the PI’s PJ34 (3μM) and EB47 (45μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI at equivalent concentrations. Clonogenic cytotoxicity assays showed that CI syndrome cells exhibit between 30–75% cell survival compared with 100% cell survival in control cells (p<0.05) in response to PI. The homologous recombination (HR) DNA repair component, rad51 forms foci in response to DNA damage. In HR compromised cells, rad51 foci fail to form. In response to PI, immunofluorescent studies show that CI syndrome cells demonstrate severely reduced rad51 foci formation (<5%) compared to control cells (15%). This confirms that PI targets the HR deficiencies in CI syndrome cells. Histone γH2AX, phosphorylated in response to DSB had greatly increased foci formation in CI syndrome cells compared to control cells as a result of unrepaired DNA damage (25.3 vs 9.3%)(p<0.05). CI syndromes have increased transformation potential to the MDS and AML. Addition of 3μM PJ34 to the myelomonocytoid leukaemic/myelodysplastic cell line, P39 exhibited significant apoptosis, with a cell survival fraction of 65% compared to 100% in control cells (p<0.01). Immunofluorescent studies revealed reduced rad51 foci formation (6.3 vs 15%) and increased γH2AX foci formation (17.6 vs 9.3%)(p<0.01). Strikingly, we were also able to reproduce similar PI responses in the Jurkat T-cell leukaemic cell line. We next explored the use of PI in combination with DNMTi or HDACi. Whilst 3μM PJ34 offered only additive effects on decitabine cytotoxicity, a sub-optimal concentration (1μM) of PJ34 behaved synergistically with HDACi potentiating the cytotoxic effect of 200nM MS275 by 55% compared to MS275 alone (p<0.05) in P39 cells. In conclusion, we have shown that in a panel of CI syndrome and leukaemic cells, PI demonstrates significant cytotoxic responses. We also show that PI acts synergistically in combination with HDACi. Parp inhibitors can potentially exploit DSB repair defects in leukaemic cells paving the way for a targeted therapy for MDS and leukaemia.

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XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.18.6.3563 ◽

1998 ◽

Vol 18 (6) ◽

pp. 3563-3571 ◽

Cited By ~ 606

Author(s):

Murielle Masson ◽

Claude Niedergang ◽

Valérie Schreiber ◽

Sylviane Muller ◽

Josiane Menissier-de Murcia ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Mammalian Cells ◽

Excision Repair ◽

Cell Cycle Checkpoint ◽

Dna Breaks ◽

Protective Function ◽

C Terminus ◽

Base Excision

ABSTRACT Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30 ) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in theSaccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.

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Circadian Rhythm of NER and ATR Pathways

Biomolecules ◽

10.3390/biom11050715 ◽

2021 ◽

Vol 11 (5) ◽

pp. 715

Author(s):

Tae-Hong Kang

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Circadian Rhythm ◽

Circadian Clock ◽

Uv Radiation ◽

Mammalian Cells ◽

Cell Cycle Checkpoint ◽

Dna Damage Responses ◽

Cycle Checkpoint

Genomic integrity is constantly insulted by solar ultraviolet (UV) radiation. Adaptative cellular mechanisms called DNA damage responses comprising DNA repair, cell cycle checkpoint, and apoptosis, are believed to be evolved to limit genomic instability according to the photoperiod during a day. As seen in many other key cellular metabolisms, genome surveillance mechanisms against genotoxic UV radiation are under the control of circadian clock systems, thereby exhibiting daily oscillations in their catalytic activities. Indeed, it has been demonstrated that nucleotide excision repair (NER), the sole DNA repair mechanism correcting UV-induced DNA photolesions, and ataxia–telangiectasia-mutated and Rad3-related (ATR)-mediated cell cycle checkpoint kinase are subjected to the robust control of the circadian clock. The molecular foundation for the circadian rhythm of UV-induced DNA damage responses in mammalian cells will be discussed.

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HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

Nature Communications ◽

10.1038/s41467-021-21302-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fa-Hui Sun ◽

Peng Zhao ◽

Nan Zhang ◽

Lu-Lu Kong ◽

Catherine C. L. Wong ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Active Site ◽

Negative Charge ◽

Structural Data ◽

Dna Breaks ◽

Adp Ribosylation ◽

Local Conformation ◽

Key Residues ◽

Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

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The Interactions of DNA Repair, Telomere Homeostasis, and p53 Mutational Status in Solid Cancers: Risk, Prognosis, and Prediction

Cancers ◽

10.3390/cancers13030479 ◽

2021 ◽

Vol 13 (3) ◽

pp. 479

Author(s):

Pavel Vodicka ◽

Ladislav Andera ◽

Alena Opattova ◽

Ludmila Vodickova

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Dna Lesions ◽

Cell Cycle Checkpoint ◽

Solid Cancer ◽

Genomic Integrity ◽

Repair Capacity ◽

Mutational Status ◽

Telomere Homeostasis

The disruption of genomic integrity due to the accumulation of various kinds of DNA damage, deficient DNA repair capacity, and telomere shortening constitute the hallmarks of malignant diseases. DNA damage response (DDR) is a signaling network to process DNA damage with importance for both cancer development and chemotherapy outcome. DDR represents the complex events that detect DNA lesions and activate signaling networks (cell cycle checkpoint induction, DNA repair, and induction of cell death). TP53, the guardian of the genome, governs the cell response, resulting in cell cycle arrest, DNA damage repair, apoptosis, and senescence. The mutational status of TP53 has an impact on DDR, and somatic mutations in this gene represent one of the critical events in human carcinogenesis. Telomere dysfunction in cells that lack p53-mediated surveillance of genomic integrity along with the involvement of DNA repair in telomeric DNA regions leads to genomic instability. While the role of individual players (DDR, telomere homeostasis, and TP53) in human cancers has attracted attention for some time, there is insufficient understanding of the interactions between these pathways. Since solid cancer is a complex and multifactorial disease with considerable inter- and intra-tumor heterogeneity, we mainly dedicated this review to the interactions of DNA repair, telomere homeostasis, and TP53 mutational status, in relation to (a) cancer risk, (b) cancer progression, and (c) cancer therapy.

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