Telomere lengths in human oocytes, cleavage stage embryos and blastocysts

Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA-repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and pre-implantation embryos, by quantitative fluorescence in-situ hybridisation (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in pre-implantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 vs 8.43 vs 12.22kb respectively, p<0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of pre-implantation embryo development

The MutSβ complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair

Loss of the DNA mismatch repair (MMR) protein MSH3 leads to the development of a variety of tumors in mice without significantly affecting survival rates, suggesting a modulating role for the MutSβ (MSH2-MSH3) complex in late-onset tumorigenesis. To better study the role of MSH3 in tumor progression, we crossed Msh3(-/-) mice onto a tumor predisposing p53-deficient background. Survival of Msh3/p53 mice was not reduced compared with p53 single mutant mice; however, the tumor spectrum changed significantly from lymphoma to sarcoma, indicating MSH3 as a potent modulator of p53-driven tumorigenesis. Interestingly, Msh3(-/-) mouse embryonic fibroblasts displayed increased chromatid breaks and persistence of γH2AX foci following ionizing radiation, indicating a defect in DNA double-strand break repair (DSBR). Msh3/p53 tumors showed increased loss of heterozygosity, elevated genome-wide copy-number variation and a moderate microsatellite instability phenotype compared with Msh2/p53 tumors, revealing that MSH2-MSH3 suppresses tumorigenesis by maintaining chromosomal stability. Our results show that the MSH2-MSH3 complex is important for the suppression of late-onset tumors due to its roles in DNA DSBR as well as in DNA MMR. Further, they demonstrate that MSH2-MSH3 suppresses chromosomal instability and modulates the tumor spectrum in p53-deficient tumorigenesis and possibly has a role in other chromosomally unstable tumors as well

Long telomeres are associated with clonality in wild populations of the fissiparous starfish Coscinasterias tenuispina

7 páginas, 4 figuras, 3 tablasTelomeres usually shorten during an organism’s lifespan and have thus been used as an aging and health marker. When telomeres become sufficiently short, senescence is induced. The most common method of restoring telomere length is via telomerase reverse transcriptase activity, highly expressed during embryogenesis. However, although asexual reproduction from adult tissues has an important role in the life cycles of certain species, its effect on the aging and fitness of wild populations, as well as its implications for the long-term survival of populations with limited genetic variation, is largely unknown. Here we compare relative telomere length of 58 individuals from four populations of the asexually reproducing starfish Coscinasterias tenuispina. Additionally, 12 individuals were used to compare telomere lengths in regenerating and non-regenerating arms, in two different tissues (tube feet and pyloric cecum). The level of clonality was assessed by genotyping the populations based on 12 specific microsatellite loci and relative telomere length was measured via quantitative PCR. The results revealed significantly longer telomeres in Mediterranean populations than Atlantic ones as demonstrated by the Kruskal–Wallis test (K=24.17, significant value: P-valueo0.001), with the former also characterized by higher levels of clonality derived from asexual reproduction. Telomeres were furthermore significantly longer in regenerating arms than in non-regenerating arms within individuals (pyloric cecum tissue: Mann–Whitney test, V=299, P-valueo10− 6; and tube feet tissue Student's t= 2.28, P-value =0.029). Our study suggests that one of the mechanisms responsible for the long-term somatic maintenance and persistence of clonal populations is telomere elongation.This research was financially supported by a PhD fellowship FPI-MICINN (BES-2011-044154) (ACG), the European ASSEMBLY project (227799), the Swedish Royal Academy of Sciences (ACG) and the Spanish Government project CTM2010-22218-C02. The research was also supported by a ‘Juan de la Cierva’ contract from the Spanish Government (RPP) and by the Adlerbertska Research Foundation (HNS).Peer reviewe

Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases

Bovine telomere dynamics and the association between telomere length and productive lifespan

Average telomere length (TL) in blood cells has been shown to decline with age in a range of vertebrate species, and there is evidence that TL is a heritable trait associated with late-life health and mortality in humans. In non-human mammals, few studies to date have examined lifelong telomere dynamics and no study has estimated the heritability of TL, despite these being important steps towards assessing the potential of TL as a biomarker of productive lifespan and health in livestock species. Here we measured relative leukocyte TL (RLTL) in 1,328 samples from 308 Holstein Friesian dairy cows and in 284 samples from 38 female calves. We found that RLTL declines after birth but remains relatively stable in adult life. We also calculated the first heritability estimates of RLTL in a livestock species which were 0.38 (SE = 0.03) and 0.32 (SE = 0.08) for the cow and the calf dataset, respectively. RLTL measured at the ages of one and five years were positively correlated with productive lifespan (p < 0.05). We conclude that bovine RLTL is a heritable trait, and its association with productive lifespan may be used in breeding programmes aiming to enhance cow longevity

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell-cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell-cycle division are discussed

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo

Creating gesture controlled games for robot-assisted stroke rehabilitation

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