Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?

The RAD51 recombinase is a critical effector of Homologous Recombination (HR), which is an essential DNA repair mechanism for double-strand breaks. The RAD51 protein is recruited onto the DNA break by BRCA2 and forms homopolymeric filaments that invade the homologous chromatid and use it as a template for repair. RAD51 filaments are detectable by immunofluorescence as distinct foci in the cell nucleus, and their presence is a read out of HR proficiency. RAD51 is an essential gene, protecting cells from genetic instability. Its expression is low and tightly regulated in normal cells and, contrastingly, elevated in a large fraction of cancers, where its level of expression and activity have been linked with sensitivity to genotoxic treatment. In particular, BRCA-deficient tumors show reduced or obliterated RAD51 foci formation and increased sensitivity to platinum salt or PARP inhibitors. However, resistance to treatment sets in rapidly and is frequently based on a complete or partial restoration of RAD51 foci formation. Consequently, RAD51 could be a highly valuable therapeutic target. Here, we review the multiple levels of regulation that impact the transcription of the RAD51 gene, as well as the post-translational modifications that determine its expression level, recruitment on DNA damage sites and the efficient formation of homofilaments. Some of these regulation levels may be targeted and their impact on cancer cell survival discussed.

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Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line

DNA Repair ◽

10.1016/j.dnarep.2009.05.004 ◽

2009 ◽

Vol 8 (8) ◽

pp. 886-900 ◽

Cited By ~ 28

Author(s):

Oliver Zschenker ◽

Avanti Kulkarni ◽

Douglas Miller ◽

Gloria. E. Reynolds ◽

Marine Granger-Locatelli ◽

...

Keyword(s):

Cell Line ◽

Cancer Cell ◽

Human Cancer ◽

Cancer Cell Line ◽

Double Strand Breaks ◽

Human Cancer Cell ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Increased Sensitivity ◽

Subtelomeric Regions

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Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass

Cancers ◽

10.3390/cancers12102848 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2848

Author(s):

Nicole A. Wilkinson ◽

Katherine S. Mnuskin ◽

Nicholas W. Ashton ◽

Roger Woodgate

Keyword(s):

Dna Damage ◽

Cancer Progression ◽

Chemotherapeutic Agents ◽

Tumor Formation ◽

Human Cells ◽

Double Strand Breaks ◽

Template Switching ◽

Post Translational Modifications ◽

Strand Breaks ◽

Exogenous Factors

Many endogenous and exogenous factors can induce genomic instability in human cells, in the form of DNA damage and mutations, that predispose them to cancer development. Normal cells rely on DNA damage bypass pathways such as translesion synthesis (TLS) and template switching (TS) to replicate past lesions that might otherwise result in prolonged replication stress and lethal double-strand breaks (DSBs). However, due to the lower fidelity of the specialized polymerases involved in TLS, the activation and suppression of these pathways must be tightly regulated by post-translational modifications such as ubiquitination in order to limit the risk of mutagenesis. Many cancer cells rely on the deregulation of DNA damage bypass to promote carcinogenesis and tumor formation, often giving them heightened resistance to DNA damage from chemotherapeutic agents. In this review, we discuss the key functions of ubiquitin and ubiquitin-like proteins in regulating DNA damage bypass in human cells, and highlight ways in which these processes are both deregulated in cancer progression and might be targeted in cancer therapy.

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RAD51 localization and activation following DNA damage

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1368 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 87-93 ◽

Cited By ~ 74

Author(s):

Madalena Tarsounas ◽

Adelina A. Davies ◽

Stephen C. West

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Critical Role ◽

S Phase ◽

Double Strand Breaks ◽

Focus Formation ◽

Rad51 Protein ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Binding Level

The efficient repair of double–strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA–damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.

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Tails and cuts: the role of histone post-translational modifications in the formation of programmed double-strand breaks

Biochimie ◽

10.1016/j.biochi.2004.11.017 ◽

2005 ◽

Vol 87 (7) ◽

pp. 603-612 ◽

Cited By ~ 2

Author(s):

Christophe de La Roche Saint-André

Keyword(s):

Double Strand Breaks ◽

Post Translational Modifications ◽

Strand Breaks

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Multiple roles of DNA2 nuclease/helicase in DNA metabolism, genome stability and human diseases

Nucleic Acids Research ◽

10.1093/nar/gkz1101 ◽

2019 ◽

Vol 48 (1) ◽

pp. 16-35 ◽

Cited By ~ 10

Author(s):

Li Zheng ◽

Yuan Meng ◽

Judith L Campbell ◽

Binghui Shen

Keyword(s):

Dna Replication ◽

Genome Stability ◽

Multiple Roles ◽

Tumor Promoter ◽

Genome Maintenance ◽

Post Translational Modifications ◽

Strand Breaks ◽

Cancer Cell Survival ◽

Dna Replication And Repair ◽

End Resection

Abstract DNA2 nuclease/helicase is a structure-specific nuclease, 5′-to-3′ helicase, and DNA-dependent ATPase. It is involved in multiple DNA metabolic pathways, including Okazaki fragment maturation, replication of ‘difficult-to-replicate’ DNA regions, end resection, stalled replication fork processing, and mitochondrial genome maintenance. The participation of DNA2 in these different pathways is regulated by its interactions with distinct groups of DNA replication and repair proteins and by post-translational modifications. These regulatory mechanisms induce its recruitment to specific DNA replication or repair complexes, such as DNA replication and end resection machinery, and stimulate its efficient cleavage of various structures, for example, to remove RNA primers or to produce 3′ overhangs at telomeres or double-strand breaks. Through these versatile activities at replication forks and DNA damage sites, DNA2 functions as both a tumor suppressor and promoter. In normal cells, it suppresses tumorigenesis by maintaining the genomic integrity. Thus, DNA2 mutations or functional deficiency may lead to cancer initiation. However, DNA2 may also function as a tumor promoter, supporting cancer cell survival by counteracting replication stress. Therefore, it may serve as an ideal target to sensitize advanced DNA2-overexpressing cancers to current chemo- and radiotherapy regimens.

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Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks

Cells ◽

10.3390/cells9071699 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1699 ◽

Cited By ~ 1

Author(s):

Lanni Aquila ◽

Boyko S. Atanassov

Keyword(s):

Double Strand Breaks ◽

Deubiquitinating Enzymes ◽

Dna Double Strand Breaks ◽

End Joining ◽

Comprehensive Overview ◽

Post Translational Modifications ◽

Strand Breaks ◽

Cellular Processes ◽

Downstream Effectors ◽

Non Homologous End Joining

Eukaryotic cells are constantly exposed to both endogenous and exogenous stressors that promote the induction of DNA damage. Of this damage, double strand breaks (DSBs) are the most lethal and must be efficiently repaired in order to maintain genomic integrity. Repair of DSBs occurs primarily through one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these pathways is in part regulated by histone post-translational modifications (PTMs) including ubiquitination. Ubiquitinated histones not only influence transcription and chromatin architecture at sites neighboring DSBs but serve as critical recruitment platforms for repair machinery as well. The reversal of these modifications by deubiquitinating enzymes (DUBs) is increasingly being recognized in a number of cellular processes including DSB repair. In this context, DUBs ensure proper levels of ubiquitin, regulate recruitment of downstream effectors, dictate repair pathway choice, and facilitate appropriate termination of the repair response. This review outlines the current understanding of histone ubiquitination in response to DSBs, followed by a comprehensive overview of the DUBs that catalyze the removal of these marks.

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The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00066-12 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3216-3223 ◽

Cited By ~ 20

Author(s):

Tina Manzhu Kang ◽

Jessica Yuan ◽

Angelyn Nguyen ◽

Elinne Becket ◽

Hanjing Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Anthracis ◽

Gene Knockout ◽

Aminoglycoside Antibiotic ◽

Double Strand Breaks ◽

Repair Capacity ◽

Content Type ◽

Strand Breaks ◽

Knockout Mutants ◽

Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

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Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8037 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8037-8050 ◽

Cited By ~ 12

Author(s):

J Halbrook ◽

M F Hoekstra

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Gene ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Intrachromosomal Recombination ◽

Spontaneous Frequency ◽

Type Strains

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.

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PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia

Blood ◽

10.1182/blood.v124.21.2183.2183 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2183-2183

Author(s):

Masatoshi Takagi ◽

Jiuhua Piao ◽

Takahiro Kamiya ◽

Mitsuko Masutani ◽

Shuki Mizutani

Keyword(s):

Cell Death ◽

Biological Effects ◽

Parp Inhibitor ◽

Double Strand Breaks ◽

Parp Inhibition ◽

Breast Cancers ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Daudi Cell ◽

Increased Sensitivity

Abstract Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor. Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting. Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity. Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia. Disclosures No relevant conflicts of interest to declare.

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