Biološki učinci ionizirajućega zračenja (IZ) pripisuju se oštećenjima DNA i indirektnim učincima kroz povećanu proizvodnju reaktivnih vrsta kisika. Iako se telomere rabe kao pokazatelji ...radioosjetljivosti, o njihovu ponašanju kao odgovoru na ionizirajuće zračenje u uvjetima profesionalne izloženosti i dalje se raspravlja. U ovom radu željeli smo istražiti duljinu i strukturu telomera u bolničkih radnika koji su profesionalno izloženi ionizirajućem zračenju te povezati te nalaze s oksidacijskim biomolekulama i kromosomskim aberacijama. Uzorci krvi izloženih ispitanika i zdravih kontrola uzeti su za analizu tijekom rutinskoga godišnjeg zdravstvenog pregleda. Osim kromosomskih aberacija, u uzorcima plazme izmjereni su i parametri oksidacijskoga stresa prooksidacijska/antioksidacijska ravnoteža (PAB), lipidna peroksidacija i 8-okso-dG, a procjena duljine i strukture telomera provedena je metodom Q-FISH na metafaznim kromosomima. Analiza kromosomskih aberacija pokazala je da od 34 ispitanika njih 14 ima kromosomske aberacije (skupina 1), a 20 nije imalo aberacije (skupina 2). Nije bilo značajne razlike u spolu ili dobi ni u duljini telomera između skupina. Međutim, incidencija lomljivih telomera bila je značajno veća u objema skupinama ispitanika izloženih IZ-u u usporedbi s kontrolnim ispitanicima. Produkti peroksidacije lipida i 8-okso-dG također su bili značajno viši u objema skupinama. Učestalost lomljivih telomera u pozitivnoj je korelaciji (statistički značajna) s razinama 8-okso-dG u plazmi, što sugerira da kontinuirano izlaganje niskim dozama ionizirajućeg zračenja izaziva oksidacijsko oštećenje baza, koje bi moglo biti uzrok lomljivosti telomera u profesionalno izloženih osoba. Međutim, potrebna su daljnja istraživanja kako bi se razjasnila uloga lomljivih telomera kao potencijalnih biomarkera za izloženost niskim dozama ionizirajućeg zračenja.
Ionising radiation damages DNA directly and indirectly through increased production of reactive oxygen species. Although telomeres have been reported as indicators of radiosensitivity, their ...maintenance in response to occupational exposure to low radiation doses is still a matter of debate. In this work we aimed to investigate telomere length and structure in hospital workers occupationally exposed to X-rays and to relate these findings to oxidation of biomolecules and chromosome aberrations. Blood samples of exposed participants and matching controls were taken during periodical check-ups. Chromosome aberrations and telomere length and structure were analysed in peripheral blood lymphocytes using Q-FISH, whereas oxidative stress parameters pro/antioxidant balance (PAB), lipid peroxidation, and 8-oxo-dG were measured in plasma samples. Based on the CA findings we divided the exposed group into two subgroups, of which one had chromosome aberrations in the first division metaphases and the other did not. There was no significant difference in telomere length between any of the groups. However, both subgroups showed significantly higher rate of fragile telomeres and higher lipid peroxidation product and 8-oxo-dG levels than controls. The rate of fragile telomeres significantly correlated with plasma levels of 8-oxo-dG, which suggests that continuous exposure to low radiation doses induces oxidative base damage of guanine resulting in telomere fragility.
Telomere length is considered as a biomarker of ageing, resulting in shortening during the process. The present investigation was an attempt to determine the relative telomere length in mechanical ...workshop workers. Telomere length shortening in cells during occupational exposure causes accelerated ageing. Genomic DNA was isolated from buccal epithelial cells collected from 240 individuals, comprising two groups of 120 exposed workers and 120 unexposed controls. Telomere length was measured by using real time PCR. Both telomere (T) and single copy gene (S) specific primers were used to compute the relative T/S ratio and expressed as the relative telomere length. Telomere length differed significantly between the workers and controls (p<0.05). The results showed an indirect and significant association (r=-0.356, p=0.001) between age and telomere length in the workers. This study showed that the difference in telomere length shortening was statistically significant (p<0.05) between the workers and controls. It was concluded that occupational exposure acts as a risk factor to enhance telomere length shortening and accelerate ageing.
Dužina telomera smatra se biomarkerom starenja i tokom ovog procesa rezultira skraćenjem. Ovo istraživanje predstavlja pokušaj određivanja relativne dužine telomera kod radnika u mehaničarskim radionicama. Skraćenje dužine telomera u ćelijama tokom profesionalne izloženosti izaziva ubrzano starenje. Genomska DNK izolovana je iz epitelnih ćelija unutrašnje strane obraza sakupljenih od 240 osoba, koji su činili dve grupe: 120 profesionalno izloženih radnika i 120 neizloženih kontrolnih subjekata. Dužina telomera izmerena je tehnikom PCR u realnom vremenu. Specifični prajmeri telomera (T) i gena prisutnih u jednoj kopiji (S) upotrebljeni su za izračunavanje relativne razmere T/S i izraženi kao relativna dužina telomera. Dužina telomera značajno se razlikovala između radnika i kontrolnih subjekata (p<0,05). Rezultati su pokazali da između starosti i dužine telomera kod radnika postoji indirektna i značajna povezanost (r=-0,356, p=0,001). Ova studija je pokazala da je razlika u skraćenju dužine telomera između radnika i kontrolnih subjekata bila statistički značajna (p<0,05). Zaključeno je da profesionalna izloženost predstavlja faktor rizika za znatnije skraćenje dužine telomera i ubrzano starenje.
Telomere kontroliraju stanično starenje Rubelj, Ivica
Rad Hrvatske akademije znanosti i umjetnosti (1991). Medicinske znanosti,
12/2010
508=35
Journal Article
Open access
Dobro je poznato da razni stanični tipovi sisavaca imaju ograničen rast kad se uzgajaju u kulturi, što nazivamo staničnim starenjem. Brojni dokazi upućuju na to da je takav ograničen stanični rast ...osnova procesa starenja. Nedavna otkrića pokazala su da su telomere ključne za navedene procese i za kontrolu staničnog ciklusa, (ne)stabilnosti genoma i imortalizaciju. U ovom pregledu bit će riječi o telomerama koje su pružile objašnjenja za razne fenomene procesa starenja i karcinogeneze.
Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non‐telomeric sequences in Tetrahymena, forming de novo ...telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non‐telomeric DNA oligonucleotide primers, consisting of natural telomere‐adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for non‐telomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT…s was the predominant initial DNA sequence added by telomerase in vitro onto the 3′ end of the non‐telomeric primers. The 3′ and 5′ sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double‐stranded primers with overhangs of various lengths showed a requirement for a minimal 3′ overhang of 20 nucleotides. With fully single‐stranded non‐telomeric primers, primer length up to ∼30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non‐telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non‐telomeric sequences.
DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to ...poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified ...in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.
Maintenance of telomeres--specialized complexes that protect the ends of chromosomes--is undertaken by the enzyme complex telomerase, which is a key factor that is activated in more than 80% of ...cancer cells that have been examined so far, but is absent in most normal cells. So, targeting telomere-maintenance mechanisms could potentially half tumour growth across a broad spectrum of tumour types, with little cytotoxic effect outside tumours. Here, we describe the current understanding of telomere biology, and the application of this knowledge to the development of anticancer drugs.
Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast ...chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.
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