Snaps and mends: DNA breaks and chromosomal translocations

Integrity in entirety is the preferred state of any organism. The temporal and spatial integrity of the genome ensures continued survival of a cell. DNA breakage is the first step towards creation of chromosomal translocations. In this review, we highlight the factors contributing towards the breakage of chromosomal DNA. It has been well-established that the structure and sequence of DNA play a critical role in selective fragility of the genome. Several non-B-DNA structures such as Z-DNA, cruciform DNA, G-quadruplexes, R loops and triplexes have been implicated in generation of genomic fragility leading to translocations. Similarly, specific sequences targeted by proteins such as Recombination Activating Genes and Activation Induced Cytidine Deaminase are involved in translocations. Processes that ensure the integrity of the genome through repair may lead to persistence of breakage and eventually translocations if their actions are anomalous. An insufficient supply of nucleotides and chromatin architecture may also play a critical role. This review focuses on a range of events with the potential to threaten the genomic integrity of a cell, leading to cancer.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

Zinc supplementation reduced DNA breaks in Ethiopian women.

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual's zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry. Cells from whole blood at the baseline and end point of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (P< .005), indicating a decrease in DNA strand breaks in zinc-supplemented individuals. These findings demonstrated that the comet assay could be used as a functional assay to assess the effects of zinc supplementation on DNA integrity in samples collected in a field setting where food sources of bioavailable zinc are limited. Furthermore, the comet assay was sufficiently sensitive to detect changes in zinc status as a result of supplementation despite no significant changes in plasma zinc.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

THE ROLE OF E2F1 IN THE RESPONSE TO DNA DOUBLE STRAND BREAKS

The importance of E2F transcription factors in the processes of proliferation and apoptosis are well established. E2F1, but not other E2F family members, is also phosphorylated and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also relocalizes and forms foci at sites of DNA double-strand breaks but the function of E2F1 at sites of damage is still unknown. Here I reveal that E2F1 deficiency leads to increased spontaneous DNA break and impaired recovery following exposure to ionizing radiation. In response to DNA double-strand breaks, NBS1 phosphorylation and foci formation are defective in cells lacking E2F1, but NBS1 expression levels are unaffected. Moreover, it was observed that an association between NBS1 and E2F1 is increased in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficient cells also display impaired foci formation of RPA and Rad51, which suggests a defect in DNA end resection and formation of single-stranded DNA at DNA double-strand breaks. I also found E2F1 status affects foci formation of the histone acetyltransferase GCN5 in response to DNA double-strand breaks. E2F1 is phosphorylated at serine 31 (serine 29 in mouse) by the ATM kinase as part of the DNA damage response. To investigate the importance of this event, our lab developed an E2F1 serine 29 mutant mouse model. I find that E2F1 serine 29 mutant cells show loss of E2F1 foci formation in response to DNA double-strand breaks. Furthermore, DNA repair and NBS1 foci formation are impaired in E2f1S29A/S29A cells. Taken together, my results indicate novel roles for E2F1 in the DNA damage response, which may directly promote DNA repair and genome maintenance.

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.

Chemotherapeutic compounds targeting the DNA double-strand break repair pathways : the good, the bad, and the promising

The repair of DNA double-strand breaks (DSBs) is a critical cellular mechanism that exists to ensure genomic stability. DNA DSBs are the most deleterious type of insult to a cell’s genetic material and can lead to genomic instability, apoptosis, or senescence. Incorrectly repaired DNA DSBs have the potential to produce chromosomal translocations and genomic instability, potentially leading to cancer. The prevalence of DNA DSBs in cancer due to unregulated growth and errors in repair opens up a potential therapeutic window in the treatment of cancers. The cellular response to DNA DSBs is comprised of two pathways to ensure DNA breaks are repaired: homologous recombination and non-homologous end joining. Identifying chemotherapeutic compounds targeting proteins involved in these DNA repair pathways has shown promise as a cancer therapy for patients, either as a monotherapy or in combination with genotoxic drugs. From the beginning, there have been a number of chemotherapeutic compounds that have yielded successful responses in the clinic, a number that have failed (CGK-733 and iniparib), and a number of promising targets for future studies identified. This review looks in detail at how the cell responds to these DNA DSBs and investigates the chemotherapeutic avenues that have been and are currently being explored to target this repair process.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats

Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing. (C) 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.

Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.

Regulation of DNA Double Strand Break Response

To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.

The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.

A glycine-arginine domain in control of the human MRE11 DNA repair protein.

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.