DNA double-strand breaks: Their production, recognition, and repair in eukaryotes

doi:10.1016/j.mrfmmm.2009.06.010

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 669, Issues 1–2, 2 October 2009, Pages 8-12

https://doi.org/10.1016/j.mrfmmm.2009.06.010 Get rights and content

Abstract

Human cells accumulate at least 10,000 DNA lesions every day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Among the various types of damage which can be expressed in a cell, DNA double-strand breaks (DSBs) represent the most serious threat. Different kinds of physical, chemical, and biological factors have been reported to induce DNA lesions, including DSBs. The aim of this review is to provide a basic understanding and overview of how DSBs are produced, recognized and repaired, and to describe the role of some of the genes and proteins involved in DSB repair.

Introduction

In contrast to DNA double-strand breaks (DSBs), DNA single-strand breaks (SSBs) are frequent endogenous DNA lesions which are found in human cells (10⁴ per cell per day) [1]. SSBs can be induced directly by free radicals, or more commonly as a consequence of the repair of apurinic sites generated by the depurination or repair of deaminated cytosine or other damaged bases [1]. In normal human cells, it is estimated that approximately 1% of DNA single-strand lesions are converted to approximately 50 endogenous DSBs per cell per cell cycle [2]. This number is similar to the estimate of the number of exogenous DSBs produced by 1.5–2.0 Gy of ionizing radiation (IR). Although endogenous DSBs are usually repaired with high fidelity, errors in their repair can contribute significantly to the rate of cancer in humans.

Section snippets

DNA damaging agents and conditions

Several factors and pathways are involved in DSB production (Fig. 1). The presence of γH2AX (histone H2AX which is phosphorylated at serine 139, located in the carboxy terminal tail) is accepted as a specific indicator for the presence of DSBs [3], [4]. Using antibodies to detect the presence of nuclear γH2AX foci, it was observed that these foci (and their accompanying DSBs) were seen, not only after exposure to IR (IR also acts via reactive oxygen species), but also after exposure to

Relaxing the heterochromatin at the first step

Especially in higher eukaryotic cells, some part of chromatin is condensed to make heterochromatin. It is important to relax the heterochromatin in some way in order to repair DSBs in this region. Heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin through INO80-γH2AX interaction [33]. Knockdown of the transcriptional repressor Krüppel-associated box-associated protein (KAP)-1, an ATM

Pathways of DSB repair

Failure to repair DNA lesions such as DSBs can lead to mutations, genomic instability, and cell death. Due to the severe consequences of DSBs, cells have developed two major repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) [48].

Mechanisms leading to chromosome translocations have been the focus of intense studies over the years. The importance of DSB repair pathways in genome maintenance is underscored by the fact that genomic instability is a characteristic

Conclusion

New factors and pathways with the ability to recognize and repair DSBs are being discovered and studied. DSB production is now recognized as a general occurrence in cells, and these lesions frequently arise through endogenous and exogenous events. As a consequence of evolution from prokaryotes to eukaryotes, cells have developed complex mechanisms which can recognize and repair this type of severe damage rapidly and correctly. Cells have been exposed to many types of environmental stresses, and

Conflict of interest

None.

Acknowledgements

This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also funded in part by a grant from the Central Research Institute of the Electric Power Industry of Japan, and by a grant for Exploratory Research for Space Utilization from the Japan Space Forum.

References (78)

O. Fernandez-Capetillo et al.
H2AX: the histone guardian of the genome
DNA Repair
(2004)
A. Takahashi et al.
Does γH2AX foci formation depend on the presence of DNA double strand breaks?
Cancer Lett.
(2005)
E.M. Hammond et al.
ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation
J. Biol. Chem.
(2003)
J.S. Liu et al.
Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents
Mutat. Res.
(2003)
A.J. Percy et al.
The measurement of DNA strand breaks in rat colonic mucosa by fluorometric analysis of DNA unwinding
Toxicol. Lett.
(1991)
K. Elmroth et al.
Cleavage of cellular DNA by calicheamicin γ1
DNA Repair
(2003)
H.E. Mischo et al.
Actinomycin D induces histone γ-H2AX foci and complex formation of γ-H2AX with Ku70 and nuclear DNA helicase II
J. Biol. Chem.
(2005)
M. Crul et al.
DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin
Biochem. Pharmacol.
(2003)
I.M. Ward et al.
Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress
J. Biol. Chem.
(2001)
E.M. Hammond et al.
Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest
Mutat. Res.
(2003)

E.P. Rogakou et al.

Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139

J. Biol. Chem.

(2000)

L.Y. Hao et al.

Phosphorylation of H2AX at short telomeres in T cells and fibroblasts

J. Biol. Chem.

(2004)

H. Kaneko et al.

Heat shock induces phosphorylation of histone H2AX in mammalian cells

Biochem. Biophys. Res. Commun.

(2005)

A. Takahashi et al.

Heat induces γH2AX foci formation in mammalian cells

Mutat. Res.

(2008)

A.J. Morrison et al.

INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair

Cell

(2004)

A.A. Goodarzi et al.

ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin

Mol. Cell

(2008)

M. Stucki et al.

MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks

Cell

(2005)

J.S. Kim et al.

Specific recruitment of human cohesin to laser-induced DNA damage

J. Biol. Chem.

(2002)

T. Helleday et al.

DNA double-strand break repair: from mechanistic understanding to cancer treatment

DNA Repair

(2007)

J.C. Game et al.

A genetic study of x-ray sensitive mutants in yeast

Mutat. Res.

(1974)

A. Dudas et al.

DNA double-strand break repair by homologous recombination

Mutat. Res.

(2004)

A.A. Davies et al.

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein

Mol. Cell

(2001)

M.F. Lavin

The Mre11 complex and ATM: a two-way functional interaction in recognising and signaling DNA double strand breaks

DNA Repair

(2004)

S. Agarwal et al.

DNA double-strand break repair and chromosome translocations

DNA Repair

(2006)

M.K. Shivji et al.

DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2

DNA Repair

(2004)

A. Smogorzewska et al.

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Cell

(2007)

T. Taniguchi et al.

Molecular pathogenesis of Fanconi anemia: recent progress

Blood

(2006)

S. Hussain et al.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

DNA Repair

(2006)

J. Thacker

The RAD51 gene family, genetic instability and cancer

Cancer Lett.

(2005)

E. Weterings et al.

The mechanism of non-homologous end-joining: a synopsis of synapsis

DNA Repair

(2004)

S. Thode et al.

A novel pathway of DNA end-to-end joining

Cell

(1990)

D. Buck et al.

Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly

Cell

(2006)

P. Ahnesorg et al.

XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining

Cell

(2006)

M. Audebert et al.

Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining

J. Biol. Chem.

(2004)

F. Windhofer et al.

Marked dependence on growth state of backup pathways of NHEJ

Int. J. Radiat. Oncol. Biol. Phys.

(2007)

W. Wu et al.

Repair of radiation induced DNA double strand breaks by backup NHEJ is enhanced in G₂

DNA Repair

(2008)

T. Lindahl

Instability and decay of the primary structure of DNA

Nature

(1993)

M.M. Vilenchik et al.

Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer

Proc. Natl. Acad. Sci. U.S.A.

(2003)

J.P. Banath et al.

Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks

Cancer Res.

(2003)

Cited by (84)

Zebrafish as an in vivo screening tool to establish PARP inhibitor efficacy
2021, DNA Repair
Double strand break (DSB) repair through Homologous Recombination (HR) is essential in maintaining genomic stability of the cell. Mutations in the HR pathway confer an increased risk for breast, ovarian, pancreatic and prostate cancer. PARP inhibitors (PARPi) are compounds that specifically target tumours deficient in HR. Novel PARPi are constantly being developed, but research is still heavily focussed on in vitro data, with mouse xenografts only being used in late stages of development. There is a need for assays that can: 1) provide in vivo data, 2) early in the development process of novel PARPi, 3) provide fast results and 4) at an affordable cost. Here we propose a combination of in vivo zebrafish assays to accurately quantify PARP inhibitor efficacy. We showed that PARPi display functional effects in zebrafish, generally correlating with their PARP trapping capacities. Furthermore, we displayed how olaparib mediated radiosensitization is conserved in our zebrafish model. These assays could aid the development of novel PARPi by providing early in vivo data. In addition, using zebrafish allows for high-throughput testing of combination therapies in search of novel treatment strategies.
Pt(IV) pro-drugs with an axial HDAC inhibitor demonstrate multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance in A2780/A2780cis cells
2020, Journal of Inorganic Biochemistry
Citation Excerpt :
When the DNA damage leads to double strand breaks (DSBs), Ser-139 of the histone H2AX is rapidly phosphorylated at the site of the DSB resulting in the formation of discrete foci. The γ-H2AX foci can be detected and quantified by immunofluorescence [52–54]. The number of γ-H2AX foci is directly proportional to the DSBs and is an indicator of DNA repair efficacy.
Epigenetic agents such as histone deacetylase (HDAC) inhibitors are widely investigated for use in combined anticancer therapy and the co-administration of Pt drugs with HDAC inhibitors has shown promise for the treatment of resistant cancers. Coordination of an HDAC inhibitor to an axial position of a Pt(IV) derivative of cisplatin allows the combination of the epigenetic drug and the Pt chemotherapeutic into a single molecule. In this work we carry out mechanistic studies on the known Pt(IV) complex cis,cis,trans-[Pt(NH₃)₂Cl₂(PBA)₂] (B) with the HDAC inhibitor 4-phenylbutyrate (PBA) and its derivatives cis,cis,trans-[Pt(NH₃)₂Cl₂(PBA)(OH)] (A), cis,cis,trans-[Pt(NH₃)₂Cl₂(PBA)(Bz)] (C), and cis,cis,trans-[Pt(NH₃)₂Cl₂(PBA)(Suc)] (D) (Bz = benzoate, Suc = succinate). The comparison of the cytotoxicity, effect on HDAC activity, reactive oxygen species (ROS) generation, γ-H2AX (histone 2A-family member X) foci generation and induction of apoptosis in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells shows that A – C exhibit multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance.
DNA mismatch repair and its many roles in eukaryotic cells
2017, Mutation Research - Reviews in Mutation Research
DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1–independent subpathways of MMR is not known. This review summarizes recent literature on eukaryotic MMR, with emphasis on the diverse cellular roles of eukaryotic MMR proteins, the mechanism of strand discrimination and cross-talk/interactions between and co-regulation of MMR and other DNA repair pathways in eukaryotic cells. The main conclusion of the review is that MMR proteins contribute to genome stability through their ability to recognize and promote an appropriate cellular response to aberrant DNA structures, especially when they arise during DNA replication. Although the molecular mechanism of MMR in the eukaryotic cell is still not completely understood, increased used of single-molecule analyses in the future may yield new insight into these unsolved questions.
Characteristics and Application of CRISPR/Casl2a Genome Editing Technology
2023, China Biotechnology
IN VITRO ANALYSIS OF GENOTOXIC EFFECTS OF DENTAL PANORAMIC RADIOGRAPHY: EVIDENCE OF POTENTIAL GENOTOXICITY USING THE COMET TEST
2023, Revista de Gestao Social e Ambiental
Ionizing Radiation-Induced DNA Damage Response in Primary Melanocytes and Keratinocytes of Human Skin
2022, Cytogenetic and Genome Research

View all citing articles on Scopus

View full text

Mini reviewDNA double-strand breaks: Their production, recognition, and repair in eukaryotes

Abstract

Introduction

Section snippets

DNA damaging agents and conditions

Relaxing the heterochromatin at the first step

Pathways of DSB repair

Conclusion

Conflict of interest

Acknowledgements

DNA Repair

Cancer Lett.

J. Biol. Chem.

Mutat. Res.

Toxicol. Lett.

DNA Repair

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Mutat. Res.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Mutat. Res.

Cell

Mol. Cell

Cell

J. Biol. Chem.

DNA Repair

Mutat. Res.

Mutat. Res.

Mol. Cell

DNA Repair

DNA Repair

DNA Repair

Cell

Blood

DNA Repair

Cancer Lett.

DNA Repair

Cell

Cell

Cell

J. Biol. Chem.

Int. J. Radiat. Oncol. Biol. Phys.

DNA Repair

Instability and decay of the primary structure of DNA

Nature

Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer

Proc. Natl. Acad. Sci. U.S.A.

Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks

Cancer Res.

Mini review
DNA double-strand breaks: Their production, recognition, and repair in eukaryotes