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 Table of Contents  
REVIEW ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 2  |  Page : 93-97

DNA double-strand break repair in mammals


Department of Biochemistry, n Institute of Science, Bengaluru, Karnataka, India

Date of Web Publication14-Jun-2017

Correspondence Address:
Sathees C Raghavan
Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_18_17

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  Abstract 


Failure in repair of DNA double-strand breaks (DSBs) could result in various disorders in mammals including cancer. Among various exogenous agents, radiation is one of the primary causes for induction of DSBs. Homologous recombination, nonhomologous end-joining, and a less efficient microhomology-mediated end-joining are responsible for repair of DSBs to ensure the genomic integrity and stability. This review highlights DNA damage response (DDR) induced following various insults to the genome and how the DNA repair mechanisms have evolved to restore genomic integrity. We also briefly discuss the potential therapeutic targets associated with DDR and DSB repair and novel inhibitors developed against such targets and their well-defined mechanism of action, which may increase sensitivity to traditional radio- and chemo-therapeutic modalities.

Keywords: DNA damage, double-strand breaks, genomic instability, radiation, radioprotection


How to cite this article:
Pandey M, Raghavan SC. DNA double-strand break repair in mammals. J Radiat Cancer Res 2017;8:93-7

How to cite this URL:
Pandey M, Raghavan SC. DNA double-strand break repair in mammals. J Radiat Cancer Res [serial online] 2017 [cited 2017 Dec 16];8:93-7. Available from: http://www.journalrcr.org/text.asp?2017/8/2/93/208022


  DNA Damage Top


Ionizing radiation (IR) is one of the major causes of DNA double-strand breaks (DSBs) within the genome. Besides radiation, exogenous factors such as free radicals generated by pollution, radiomimetic compounds, and pesticides also induce DSBs. Replication errors, inadvertent enzyme actions such as topoisomerase failures, class switch recombination and V(D)J recombination processes, and free radicals generated through oxidative metabolism are some of the endogenous agents that are responsible for the generation of DSBs.[1],[2],[3] DNA DSBs are considered to be the most lethal among all the lesions which, if left unrepaired or improperly repaired, can trigger cell cycle arrest, cause cell death, and may potentially induce a wide variety of genetic alterations to cause oncogenic transformations.[4],[5],[6]


  DNA Damage Response Top


The signaling networks that constitute DNA damage response (DDR), activate an elaborate and dedicated DNA repair system, and elicit cell-cycle arrest (cell-cycle checkpoint) to ensure proper repair of any transient damage. In case of severe irreparable DNA damage, DDR also helps in triggering apoptosis to completely eliminate the cells with lesions rather than allowing them to propagate the faulty genome to future generations [Figure 1].[7],[8]
Figure 1: General outline of DNA damage response and ensuing signal transduction pathway to repair DNA breaks in mammalian cells. Double-strand breaks are induced in the cells upon exposure to various endogenous and exogenous agents, which are sensed by damage sensors (g-H2AX, MRE11, Rad50 and Nbs1 complex, PARP) that in turn, trigger downstream signaling pathways (which include various transducers [ataxia telangiectasia mutated/ataxia telangiectasia mutated and Rad3] and effectors [breast cancer-associated protein 1, claspin, 53BP1]), to repair these breaks, robustly by either homologous recombination, nonhomologous end-joining, or microhomology-mediated end-joining pathways. Misrepaired breaks could result in chromosomal translocations and genomic instability or can even result in apoptosis

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On induction, DSBs are detected by damage sensors, ataxia telangiectasia mutated (ATM) and ATM-Rad3- related (ATR) ATR, which also function as a signal transducers. ATM/ATR, with the aid of mediators, breast cancer-associated protein 1 (BRCA1), claspin, 53BP1, and MDC1, transduce the signal to transducers such as checkpoint kinase-1/2, which, in turn, activate or deactivate the effectors such as p53 that directly participate in cell cycle arrest and maintain a strong crosstalk with the DNA repair machinery as well [Figure 1].[3]


  Repair of DNA Double-strand Breaks Top


To repair lethal DSBs, the mammalian cells employ two major mutually exclusive pathways, homologous recombination (HR), and nonhomologous end-joining (NHEJ), thereby maintaining genomic integrity [Figure 2].[9],[10] NHEJ is an error-prone pathway, operative throughout the cell cycle, joins DSBs with no or minimal homology, whereas HR restores the genomic sequence duplex DNA precisely in diploid organism with cells in S and G2 phases of cell cycle, by utilizing sister chromatids as template for repair.[9]
Figure 2: Schematic showing double-strand break repair pathways in mammalian cells. Homologous recombination is an error-free double-strand break repair process, involves 5' end resection of the broken ends, with Rad51 as the major player. The second pathway is nonhomologous end-joining, which initiates with KU70/80 binding at the site of double-strand breaks that undergo ligation by ligase IV/X-ray repair cross-complementing protein 4/X-ray repair cross-complementing protein 4-like factor/PAralog of X-ray repair cross-complementing protein 4 and X-ray repair cross-complementing protein 4-like factor complex. The third, alt-nonhomologous end-joining or microhomology-mediated end-joining pathway ensues by recognition of breaks with microhomology by MRE11, Rad50 and Nbs1 complex, PARP1 and possibly by Pol Q proteins, and ligated by ligase III/X-ray repair cross-complementing protein 1 complex

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  Nonhomologous DNA End-Joining Top


The first protein to bind DSBs during NHEJ is KU70/80, which acts as a platform for the recruitment of downstream proteins, such as DNA-dependent protein kinase catalytic subunit (PKcs) and artemis (which processes damaged ends) and other end-processing enzymes.[11],[12],[13] Template dependent and independent DNA synthesis step is carried out by DNA pol λ and μ polymerases, while the final step of ligation is catalyzed by a ternary complex of Ligase IV, X-ray repair cross-complementing protein 4 (XRCC4), and XRCC4-like factor (XLF).[10] Recently, a novel DSB repair protein, PAralog of XRCC4 and XLF (PAXX), that shares structural and functional homology with XRCC4 was identified.[14] It appears that PAXX is recruited to the site of DSBs in a KU-dependent manner within the cells and promotes binding of other NHEJ factors to the damaged sites [Figure 2].

NHEJ pathway is also responsible for repair of recombination-activating genes-induced DSBs during V(D)J recombination in lymphocytes, essential for the generation of antibody diversity.[15],[16]

Mouse models and genetic diseases have shown that impairment of classical NHEJ (c-NHEJ) proteins leads to immunodeficiency. Failure in VDJ recombination due to c-NHEJ dysfunction could result in growth retardation, developmental delay, etc., in B- and T-cells which confers sensitivity to IR and other DSB-inducing agents.[17],[18],[19]


  Double-strand Break Repair by Homologous Recombination Top


In diploid organisms, HR-mediated DSB repair restores the genomic DNA sequence precisely during the S and G2 phases of cell cycle, by utilizing sister chromatids as templates for repair.[9] During HR, 5' ends of DSBs are resected by a complex of MRE11, Rad50, and Nbs1 (MRN), hExo1, and CtIP proteins to generate 3' single-stranded overhangs. Rad51 binds to replication protein A (RPA). RPA-coated single-stranded DNA culminating into a complex called presynaptic filament. This is followed by strand invasion assisted by the same recombinase and its various paralogs, whereby search for homology initiates and the single strand gets base-paired with the complementary strand of the homologous chromosome to form a displacement structure called the “D-loop.” Using the complementary strand as template, single-strand DNA undergoes synthesis. Similarly, the other end is captured, the gap is filled in, and terminal ligation process occurs to mend the breaks in a highly accurate manner [Figure 2].[20] Therefore, mutations in functional HR pathway can lead to genomic instability and contribute to carcinogenesis.


  Double-strand Break Repair by Alternative Nonhomologous End-Joining/Microhomology-Mediated End-Joining Top


In the absence of key NHEJ factors, another distinct but error-prone back-up pathway known as alternative NHEJ (A-NHEJ) has been described to have an important role in DSB repair. Majority of A-NHEJ junctions utilizes microhomology at the breakpoint regions, consisting of shared sequence (ranging from 5 to 22 bp) between the two DNA ends, hence known as microhomology-mediated end-joining (MMEJ). Recent studies suggest that MMEJ is operative in both normal and cancer cells.[21],[22]

Although the machinery of MMEJ pathway is not completely defined, previous reports suggest the participation of proteins such as PARP1, Ligase III/XRCC1, Ligase I, MRN, and FEN1 in MMEJ.[23] In one of the most comprehensive studies, a novel biochemical assay system utilizing DSBs flanked by varying lengths of direct repeats was developed to investigate both enzymatic requirement and DNA sequence dependence of MMEJ.[22] Novel evidences have emphasized the indispensable role of C-terminal polymerase activity of the human polymerase POLQ (or Pol θ) in catalyzing alternative DNA end-joining in eukaryotes.[24],[25],[26] It appears that Pol θ undergoes multimerization to facilitate synapsis of the broken DNA ends (using microhomologies upto 25 bp) or sequestering free DNA ends from engaging in classical NHEJ.[26],[27] Although independent studies suggest the involvement of multiple proteins using gene knockdown or knockout strategies, their specific role in different steps is yet to be defined [Figure 2].

One remarkable pathological agent that has recently been discovered to significantly enhance MMEJ is the organochlorine pesticide, endosulfan that has known to be a persistent environmental pollutant with speculated untoward effects on humans,[28],[29] inducing intense DNA damage and genomic instability.


  Targeting Double-strand Break Repair Pathways Top


Radio- and chemo-therapy largely rely on accumulation of DNA damage in cancer cells yet are accompanied by significant damage to the surrounding normal tissues. In addition to these well-established ways for treating cancer, there is a dire need to develop targeted therapy based on different mechanisms of DDR restricted to cancer cells. DSB repair genes are known to be upregulated in many cancers, which become one of the reasons for chemo- and radio-resistance in patients. Furthermore, there are cases in which enhanced mutation rate due to compromised DNA repair results in downregulation of tumor suppressors such as BRCA1/2. Therefore, targeting DDR by specifically abrogating DSB repair in cancer cells is emerging as an attractive strategy to eliminate cancer. Reports suggest that increased activities of DNA repair proteins can predispose the cells to cancer.[30],[31] However, a defective repair pathway and the ability of synthetic compounds to inhibit or activate DNA repair can also govern a patient's response to therapy.[32]

Among the well-known DNA repair inhibitors, mirin is extensively used as biochemical inhibitor of MRE11[33] while Wortmannin is developed against DNA-PKcs as a pan PI3K inhibitor.[34] An attractive target Ligase IV has been extensively explored for its anticancer therapeutic value. A major breakthrough came in the form of a potent and specific Ligase IV inhibitor named as SCR7, which can inhibit NHEJ both in vitro and ex vivo in a ligase IV-dependent manner.[35] SCR7 served as one of the best examples of anticancer therapeutic strategy targeting DSB repair and exhibiting synergistic effects with radio- and chemo-therapeutic agents.[35] Besides, a nanomaterial-encapsulated version of SCR7 inhibited NHEJ leading to ~5-fold increase in cell death.[36] Two other compounds, SCR17 and SCR21, have been recently identified using in vitro and ex vivo assays to specifically inhibit nick ligation activity of DNA Ligase I, thus affecting replication in cancer cells.[37]

The ultimate goal in the field of cancer therapeutics is strategic synergism of repair inhibitors coupled with genotoxic agents such as ionization radiation. This will help in sensitizing cancer cells even with low dose of radiation and chemotherapeutic agents, hence reducing the side effects and overcoming the problem of resistance.

Financial support and sponsorship

MP is supported by Senior Research Fellowship from IISc, India.

Conflicts of interest

There are no conflicts of interest

 
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