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Accurate transmission of genomic information across multiple cell divisions and generations, without any losses or errors, is fundamental to all living organisms. To achieve this goal, eukaryotes devised chromosomes. Eukaryotic genomes are represented by multiple linear chromosomes in the nucleus, each carrying a centromere in the middle, a telomere at both ends, and multiple origins of replication along the chromosome arms. Although all three of these DNA elements are indispensable for chromosome function, centromeres and telomeres possess the potential to detach from the original chromosome and attach to new chromosomal positions, as evident from the events of telomere fusion, centromere inactivation, telomere healing, and neocentromere formation. These events seem to occur spontaneously in nature but have not yet been elucidated clearly, because they are relatively infrequent and sometimes detrimental. To address this issue, experimental setups have been developed using model organisms such as yeast. In this article, we review some of the key experiments that provide clues as to the extent to which these paradoxical and elusive features of chromosomally indispensable elements may become valuable in the natural context.

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Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

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The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system.

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Two distinct groups of parasitic nematodes use programmed DNA elimination to silence germline-expressed genes in the somatic cells (ascarids) or for sex determination (Strongyloides spp.). In the ascarids, DNA is lost only in pre-somatic cells during early embryogenesis, leading to a reduced somatic genome compared to the intact germ cell genome. Comparative genome analysis has provided information on the retained vs. eliminated sequences, DNA breaks, a full chromosome view on DNA elimination, and the evolutionary conservation of DNA elimination among ascarids. These studies have revealed novel insights into the functions and mechanisms of DNA elimination and provided a reference for in-depth molecular analysis of DNA elimination. Here, I describe the genomics methods we used to study programmed DNA elimination, focusing on the parasitic nematode Ascaris.

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Germline and somatic genomes are in general the same in a multicellular organism. However, programmed DNA elimination leads to a reduced somatic genome compared to germline cells. Previous work on the parasitic nematode Ascaris demonstrated that programmed DNA elimination encompasses high-fidelity chromosomal breaks and loss of specific genome sequences including a major tandem repeat of 120 bp and ~1,000 germline-expressed genes. However, the precise chromosomal locations of these repeats, breaks regions, and eliminated genes remained unknown. We used PacBio long-read sequencing and chromosome conformation capture (Hi-C) to obtain fully assembled chromosomes of Ascaris germline and somatic genomes, enabling a complete chromosomal view of DNA elimination. We found that all 24 germline chromosomes undergo comprehensive chromosome end remodeling with DNA breaks in their subtelomeric regions and loss of distal sequences including the telomeres at both chromosome ends. All new Ascaris somatic chromosome ends are recapped by de novo telomere healing. We provide an ultrastructural analysis of Ascaris DNA elimination and show that eliminated DNA is incorporated into double membrane-bound structures, similar to micronuclei, during telophase of a DNA elimination mitosis. These micronuclei undergo dynamic changes including loss of active histone marks and localize to the cytoplasm following daughter nuclei formation and cytokinesis where they form autophagosomes. Comparative analysis of nematode chromosomes suggests that chromosome fusions occurred, forming Ascaris sex chromosomes that become independent chromosomes following DNA elimination breaks in somatic cells. These studies provide the first chromosomal view and define novel features and functions of metazoan programmed DNA elimination.

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The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called "telomere healing," and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity.IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity.

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BACKGROUND: All human chromosomes are capped by tandem repeat (TTAGGG)n sequences that protect them against end-to-end fusion and are essential to chromosomal replication and integrity. Therefore, after a chromosomal breakage, the deleted chromosomes must be stabilized by retaining the telomere or acquiring a new cap, by telomere healing or telomere capture. There are few reports with molecular approaches on the mechanisms involved in stabilization of 18q terminal deletions. RESULTS: In this study we analyzed nine patients with 18q terminal deletion identified by G-banding and genomic array. FISH using PNA probe revealed telomeric signals in all deleted chromosomes tested. We fine-mapped breakpoints with customized arrays and sequenced six terminal deletion junctions. In all six deleted chromosomes sequenced, telomeric sequences were found directly attached to the breakpoints. Little or no microhomology was found at the breakpoints and none of the breaks sequenced were located in low copy repeat (LCR) regions, though repetitive elements were found around the breakpoints in five patients. One patient presented a more complex rearrangement with two deleted segments and an addition of 17 base pairs (bp). CONCLUSIONS: We found that all six deleted chromosomes sequenced were probably stabilized by the healing mechanism leading to a neotelomere formation.