Marta Martín

Shorter telomeres, accelerated ageing and increased lymphoma in DNA-PKcs-deficient mice.

Non‐homologous end joining (NHEJ) is the principal repair mechanism used by mammalian cells to cope with double‐strand breaks (DSBs) that continually occur in the genome. One of the key components of the mammalian NHEJ machinery is the DNA‐PK complex, formed by the Ku86/70 heterodimer and the DNA‐PK catalytic subunit (DNA‐PKcs). Here, we report on the detailed life‐long follow‐up of DNA‐PKcs‐defective mice. Apart from defining a role of DNA‐PKcs in telomere length maintenance in the context of the ageing organism, we observed that DNA‐PKcs‐defective mice had a shorter life span and showed an earlier onset of ageing‐related pathologies than the corresponding wild‐type littermates. In addition, DNA‐PKcs ablation was associated with a markedly higher incidence of T lymphomas and infections. In conclusion, these data link the dual role of DNA‐PKcs in DNA repair and telomere length maintenance to organismal ageing and cancer.

The effect of breed-production systems on the myosin heavy chain 1, the biochemical characteristics and the colour variables of Longissimus thoracis from seven Spanish beef cattle breeds

The effect of breed-production system on the myosin heavy chain 1 (MHC-I), the biochemical characteristics and the colour variables of longissimus thoracis (LT) from seven beef breeds was studied: Asturiana de la Montaña (AM), Asturiana de los Valles (AV), Avileña-Negra Ibérica (A-NI), Bruna dels Pirineus (BP), Morucha (MO), Pirenaica (PI) and Retinta (RE) (Age at slaughter between 368 and 541 days; carcass weight between 249 and 334 kg). Significant differences between breed-production systems were found for all traits evaluated. LT from the MO, a rustic type breed, was the most oxidative (MHC-I, 39.3%; isocitrate dehydrogenase activity, 52 nmol min(-1) mg(-1); pigment content, 188.4 μg acid haematin g(-1)) and showed a low L* value (32.6) and high a* and C* values (24 and 27.2, respectively). In terms of meat colour (L* and a*) the canonical discriminant analysis separated the breeds into two groups, the AV, the PI and the A-NI (the lightest ones) from the AM and the MO breeds (the reddest and darkest) whereas the BP showed an intermediate position. The RE and the A-NI were distinguished from the others by their high intramuscular fat content. Meat colour was affected by the muscle biochemical traits in the breed-production systems studied.

Genetic activities in micronuclei: is the DNA entrapped in micronuclei lost for the cell?

Micronuclei are good markers of genotoxic exposure in humans and their scoring has been extensively used to identify potential genotoxic agents. Micronuclei are also indicators of chromosomal instability, since the frequency of micronuclei is higher in tumour cells and cells with a defective DNA damage repair system or disrupted cell cycle checkpoint machinery. Despite the widespread use of this biomarker, information on the basic biology of micronuclei and the impact of micronuclei on the cell is relatively controversial. In some cell systems, micronuclei are considered to be genetic material that is lost for the cell; whereas other studies suggest that micronuclear DNA is actively transcribed and its genes are fully expressed. Recently, evidence has accumulated suggesting that damaged DNA entrapped in micronuclei induces a defective cell cycle checkpoint arrest and DNA repair response, and that micronuclear content can be degraded without inducing an immediate cell cycle arrest or causing the cell to enter apoptosis. Overall, these findings emphasise the important consequences of micronucleus formation in terms of chromosomal instability in general and gene loss in particular.

Shortened telomeres join to DNA breaks interfering with their correct repair

Telomeres cap chromosome ends, avoiding end-to-end fusions and subsequent chromosome instability. Telomeric functions and DNA repair pathways are closely related. Telomere dysfunction has been shown to result in hypersensitivity to ionizing radiation. In this study, we have used the telomerase knockout model to investigate how telomere shortening influences the correct repair of broken chromosomes. We show that the correct repair of double-strand breaks is impaired in telomerase knockout mice. The chromosomes with shortened telomeres fuse to radiation-induced breaks, interfering with the correct rejoining of the broken ends. This type of fusion is responsible for the increased chromosome instability observed in this mouse model, after exposure to ionizing radiation. Our finding may be important for understanding the increased radiation sensitivity associated with age in humans, as well as for comprehending the interindividual differences to the cytotoxic effects of radiation therapy in cancer patients.

DNA lesions sequestered in micronuclei induce a local defective-damage response

Micronuclei are good markers of chromosome instability and, among other disturbances, are closely related to double-strand break induction. The ability of DNA lesions sequestered in the micronuclear bodies to activate the complex damage-signalling network is highly controversial since some repair factors have not been consistently detected inside micronuclei. In order to better understand the efficiency of the response induced by micronuclear DNA damage, we have analyzed the presence of DNA damage-response factors and DNA degradation markers in these structures. Radiation-induced DNA double-strand breaks produce a modification of chromatin structural proteins, such as the H2AX histone, which is rapidly phosphorylated around the break site. Strikingly, we have been able to distinguish two different phosphoH2AX (gammaH2AX) labelling patterns in micronuclei: discrete foci, indicating DSB presence, and uniform labelling affecting the whole micronucleus, pointing to genomic DNA fragmentation. At early post-irradiation times we observed a high fraction of micronuclei displaying gammaH2AX foci. Co-localization experiments showed that only a small fraction of the DSBs in micronuclei were able to properly recruit the p53 binding protein 1 (53BP1) and the meiotic recombination 11 (MRE11). We suggest that trafficking defects through the micronuclear envelope compromise the recruitment of DNA damage-response factors. In contrast to micronuclei displaying gammaH2AX foci, we observed that micronuclei showing a gammaH2AX extensive-uniform labelling were more frequently observed at substantial post-irradiation times. By means of TUNEL assay, we proved that DNA degradation was carried out inside these micronuclei. Given this scenario, we propose that micronuclei carrying a non-repaired DSB are conduced to their elimination, thus favouring chromosome instability in terms of allele loss.

Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs-Deficient Mouse Embryonic Fibroblasts

Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNA-PKcs-/- MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs-/- cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs-/- MEFs than in their wild-type counterparts. Spectral karyotyping-fluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs-/- MEFs. Because DNA-PKcs-/- MEFs show a strong G1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs-/- MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events.

Nuclear envelope defects impede a proper response to micronuclear DNA lesions

When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.

Radiation-induced chromosome breaks in ataxia-telangiectasia cells remain open

Purpose : To determine if broken chromosome-end healing mechanisms through the addition of new telomeric sequences exist in cells having difficulties in rejoining the ends of broken chromosomes. Materials and Methods : A full-colour painting protocol of all human chromosomes by FISH was combined with a telomeric and centromeric labelling using PNA probes to characterize the rejoining pattern and telomere status of radiation-induced chromosome breaks in ataxia-telangiectasia (A-T) and normal lymphoblastoid cell lines. Results : It was first established that the cell lines used for chromosome healing analysis were chromosomally stable. FISH analysis provided evidence that the frequency of deleted chromosomes, apparently unrejoined, was much higher in A-T than in normal cells, as expected by the role of ATM in cell-cycle control, as well as in DNA repair. In spite of their high frequency, broken chromosome ends in A-T cells do not seem to act as substrates for telomerase since additional terminal telomere sequences (more than the 92 expected pairs) indicative of chromosome healing were never observed. Broken chromosome ends in A-T cells remained open. Conclusion : The disability of cells to rejoin broken chromosome ends does not lead to the healing of DSBs by the acquisition of new telomeric sequences.

ATM and DNA-PKcs make a complementary couple in DNA double strand break repair

The interplay between ATM and DNA-PKcs kinases during double strand breaks (DSBs) resolution is still a matter of debate. ATM and DNA-PKcs participate differently in the DNA damage response pathway (DDR), but important common aspects are indeed found: both of them are activated when faced with DSBs, they share common targets in the DDR and the absence of either kinase results in faulty DSB repair. Absence of ATM translates into timely repair that, nevertheless, is incomplete. On the other hand, DNA-PKcs absence translates into slower repair, which in turn gives rise to the accumulation of simple and complex reorganizations. These outcomes confirm that the function of both protein kinases is essential to guarantee genome integrity. Interestingly, V(D)J and CSR recombination events provide a powerful tool to study the interplay between both kinases in DSB repair. Although the physiological DSBs generated during V(D)J and CSR recombination are resolved by the non-homologous end-joining (NHEJ) repair pathway, ATM absence during these events translates into chromosome translocations. These results suggest that NHEJ accuracy is threatened in the absence of ATM, which may play a role in avoiding illegitimate repair by favouring the joining of the correct DNA ends. Indeed, simultaneous DNA-PKcs and ATM deficiency during V(D)J and CSR recombination translates into a synergistic increase in potentially dangerous chromosomal translocations and deletions. Although the exact nature of their interaction remains elusive, the evidence indicates that ATM and DNA-PKcs play complementary roles that allow complete and legitimate DSB repair to be reached. Faithful repair can only be achieved by the presence and correct functioning of both kinases: while DNA-PKcs ensures fast rejoining, ATM guarantees complete repair.

Impaired nuclear functions in micronuclei results in genome instability and chromothripsis

Micronuclei (MN) have generally been considered a consequence of DNA damage and, as such, have been used as markers of exposure to genotoxic agents. However, advances in DNA sequencing methods and the development of high-resolution microscopy with which to analyse chromosome dynamics in live cells have been fundamental in building a more refined view of the existing links between DNA damage and micronuclei. Here, we review recent progress indicating that defects of micronuclei affect basic nuclear functions, such as DNA repair and replication, generating massive damage in the chromatin of the MN. In addition, the physical isolation of chromosomes within MN offers an attractive mechanistic explanation for chromothripsis, a massive local DNA fragmentation that produces complex rearrangements restricted to only one or a few chromosomes. When micronuclear chromatin is reincorporated in the daughter cell nuclei, the under-replicated, damaged or rearranged micronuclear chromatin might contribute to genome instability. The traditional conception of micronuclei has been overturned, as they have evolved from passive indicators of DNA damage to active players in the formation of DNA lesions, thus unravelling previously unforeseen roles of micronuclei in the origins of chromosome instability.