Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death

Functional telomeres are protected from non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways. Replication is a critical period for telomeres because of the requirement for reconstitution of functional protected telomere conformations, a process that involves DNA repair proteins. Using knockdown of DNA-PKcs and Rad51 expression in three different cell lines, we demonstrate the respective involvement of NHEJ and HR in the formation of telomere aberrations induced by the G-quadruplex ligand 360A during or after replication. HR contributed to specific chromatid-type aberrations (telomere losses and doublets) affecting the lagging strand telomeres, whereas DNA-PKcs-dependent NHEJ was responsible for sister telomere fusions as a direct consequence of G-quadruplex formation and/or stabilization induced by 360A on parental telomere G strands. NHEJ and HR activation at telomeres altered mitotic progression in treated cells. In particular, NHEJ-mediated sister telomere fusions were associated with altered metaphase-anaphase transition and anaphase bridges and resulted in cell death during mitosis or early G1. Collectively, these data elucidate specific molecular and cellular mechanisms triggered by telomere targeting by the G-quadruplex ligand 360A, leading to cancer cell death.     

Telomere dynamics unique to meiotic prophase: formation and significance of the bouquet

Telomeres carry out conserved and possibly ancient functions in meiosis. During the specialized prophase of meiosis I, meiotic prophase, telomeres cluster on the nuclear envelope and move the diploid genetic material around within the nucleus so that homologous chromosomes can align two by two and efficiently recombine with precision. This recombination is in turn required for proper segregation of the homologs into viable haploid daughter cells. The meiosis-specific telomere clustering on the nuclear envelope defines the bouquet stage, so named for its resemblance to the stems from a bouquet of cut flowers. Here, a comparative analysis of the literature on meiotic telomeres from a variety of different species illustrates that the bouquet is nearly universal among life cycles with sexual reproduction. The bouquet has been well documented for over 100 years, but our understanding of how it forms and how it functions has only recently begun to increase. Early and recent observations document the timing and provide clues about the functional significance of these striking telomere movements.     

Telomeres and DNA double-strand breaks: ever the twain shall meet?

Telomeres were first recognized as a bona fide constituent of the chromosome based on their inability to rejoin with broken chromosome ends produced by radiation. Today, we recognize two essential and interrelated properties of telomeres. They circumvent the so-called end-replication problem faced by genomes composed of linear chromosomes, which erode from their termini with each successive cell division. Equally vital is the end-capping function that telomeres provide, which is necessary to deter chromosome ends from illicit recombination. This latter property is critical in facilitating the distinction between the naturally occurring DNA double-strand breaks (DSBs) found at chromosome ends (i.e., telomeres) and DSBs produced by exogenous agents. Here we discuss, in a brief historical narrative, key discoveries that led investigators to appreciate the unique properties of telomeres in protecting chromosome ends, and the consequences of telomere dysfunction, particularly as related to recombination involving radiation-induced DSBs. Dedication. In appreciation of his heart-felt commitment to research and education, and the life-long influence he has had on the lives of students and colleagues, the authors wish to dedicate this paper to Professor Joel S. Bedford. Received 21 May 2007; received after revision 28 June 2007; accepted 6 August 2007     

Telomeres and meiosis in health and disease

Beyond their role in replication and chromosome end capping, telomeres are also thought to function in meiotic chromosome pairing, meiotic and mitotic chromosome segregation as well as in nuclear organization. Observations in both somatic and meiotic cells suggest that the positioning of telomeres within the nucleus is highly specific and believed to be dependent mainly on telomere interactions with the nuclear envelope either directly or through chromatin interacting proteins. Although little is known about the mechanism of telomere clustering, some studies show that it is an active process. Recent data have suggested a regulatory role for telomere chromatin structure in telomere movement. This review will summarize recent studies on telomere interactions with the nuclear matrix, telomere chromatin structure and factors that modify telomere chromatin structure as related to regulation of telomere movement.     

Composition and conservation of the telomeric complex

The telomere is composed of telomeric DNA and telomere-associated proteins. Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered. In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1. Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres. In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins. Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein. Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.     

Genetic and molecular approaches to synthesis and action of the yeast killer toxin

Summary The K1 killer toxin ofSaccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.     

Gathering up meiotic telomeres: a novel function of the microtubule-organizing center

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering depends on conserved SUN and KASH domain nuclear membrane proteins, which form a complex called the linker of nucleoskeleton and cytoskeleton (LINC) and connect telomeres with the cytoskeleton. It has been thought that LINC-mediated cytoskeletal forces induce telomere clustering. However, how cytoskeletal forces induce telomere clustering is not fully understood. Recent study of fission yeast has shown that the LINC complex forms the microtubule-organizing center (MTOC) at the telomere, which has been designated as the “telocentrosome”, and that microtubule motors gather telomeres via telocentrosome-nucleated microtubules. This MTOC-dependent telomere clustering might be conserved in other eukaryotes. Furthermore, the MTOC-dependent clustering mechanism appears to function in various other biological events. This review presents an overview of the current understanding of the mechanism of meiotic telomere clustering and discusses the universality of the MTOC-dependent clustering mechanism.     

Structure and function of RecA-DNA complexes

While theE. coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently. It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure. With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.     

RecQ helicases and topoisomerases: components of a conserved complex for the regulation of genetic recombination

Maintenance of genomic stability relies on the efficient and accurate execution of DNA repair pathways, and is essential for cell viability and the prevention of cancer. Mutation of genes encoding RecQ helicases or topoisomerases gives rise to genomic instability through excessive recombination. Here, we review the recent biochemical and genetic evidence to indicate that these two classes of protein act in concert in a conserved pathway to maintain genomic stability by preventing inappropriate recombination.     

Telomeres and meiosis in health and disease

Telomeres are important segments of chromosomes that protect chromosome ends from nucleolytic degradation and fusion. At meiosis telomeres display an unprecedented behavior which involves their attachment and motility along the nuclear envelope. The movements become restricted to a limited nuclear sector during the so-called bouquet stage, which is widely conserved among species. Recent observations suggest that telomere clustering involves actin and/or microtubules, and is altered in the presence of impaired recombinogenic and chromosome related functions. This review aims to provide an overview of what is currently known about meiotic telomere attachment, dynamics and regulation in synaptic meiosis.     

Telomeres and meiosis in health and disease

Telomeres are important segments of chromosomes that protect chromosome ends from nucleolytic degradation and fusion. At meiosis telomeres display an unprecedented behavior which involves their attachment and motility along the nuclear envelope. The movements become restricted to a limited nuclear sector during the so-called bouquet stage, which is widely conserved among species. Recent observations suggest that telomere clustering involves actin and/or microtubules, and is altered in the presence of impaired recombinogenic and chromosome related functions. This review aims to provide an overview of what is currently known about meiotic telomere attachment, dynamics and regulation in synaptic meiosis.     

Telomeres and chromosomal instability

Telomeres are distinctive structures, composed of a repetitive DNA sequence and associated proteins, which enable cells to distinguish chromosome ends from DNA double-strand breaks. Telomere alterations, caused by replication-mediated shortening, direct damage or defective telomere-associated proteins, usually generate chromosomal instability, which is observed in senescence and during the immortalization process. In cancer cells, this chromosome instability could be extended by their ability to repair chromosomes and terminate in break-fusion-bridge cycles. Dysfunctional telomeres can be healed by activation of telomerase or by the alternative mechanism of telomere lengthening. Activation of such telomere maintenance mechanisms may help to preserve the integrity of chromosomes even if they play a role in chromosomal instability. This review focuses on molecular processes involved in telomere maintenance and chromosomal instability associated with dysfunctional telomeres in mammalian cells.Received 24 July 2003; received after revision 5 September 2003; accepted 11 September 2003     

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.     

Consensus phylogeny ofDictyostelium

The evolutionary relationship ofDictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated thatDictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishesDictyostelum's place in evolution between the yeastsSaccharomyces cerevisiae andSchizzosaccharomyces pombe and the wormCaenorhabditis elegans.     

Genetic characterization of the new morphological and UV-sensitive mutants in Coprinus cinereus. I. A UV-sensitive mutation rad 1 associated with elevated frequencies of mitotic and meiotic recombination

Studies on the effect of an UV-sensitive mutation, rad 1, in meiotic and mitotic recombination in Coprinus indicated that, in homozygous condition, rad 1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygous rad 1 diploid was shown to be much more sensitive to the recombinogenic effects of polyfunctional than than of mono- or non-functional alkylating agents.