Genesis of end-to-end chromosome fusions

Telomeres are DNA-protein complexes that form a protective cap at chromosome ends and provide a buffer against gradual loss DNA that occurs with every round of DNA replication. Telomere length is replenished by telomerase. Deficiency of telomerase in most human somatic cells causes telomere shortening with age. When telomeres become critically short, they uncap and can be processed as abnormal double strand breaks to generate end-to-end chromosome fusions. The resulting dicentric chromosomes may promote tumorigenesis, but they enter fusion-breakage-bridge cycles that impede the elucidation of the structure of the initial fusion event and a mechanistic understanding of their genesis. Current models for fusion of critically shortened, uncapped telomeres rely on PCR-based assays that typically capture fusion breakpoints created by ligation of two chromosome ends. We used two independent approaches that rely on distinctive features of the nematode C. elegans to study the frequency of direct end-to-end chromosome fusions in telomerase mutants: 1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusion events, and 2) unique subtelomeric sequences that allow for an unbiased, nearly exhaustive PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of initial end-to-end fusion events resulted from direct end-joining with no other rearrangements. We used three approaches to investigate complex fusion breakpoint structures: 1) physical analysis of the fusion breakpoint DNA by Southern blotting, 2) measurement of DNA copy number by microarray analysis, and 3) sequence analysis of fusion breakpoints recovered by inverse PCR. Duplications as large as two megabases were present at complex fusion breakpoints. Such events would have been missed by studies using typical PCR-based assays. Thus, duplications of various segments of the genome may be a major factor that drives end-to-end chromosome fusion and promotes tumor development

Caenorhabditis elegans POT-1 and POT-2 Repress Telomere Maintenance Pathways

Telomeres are composed of simple tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion or inappropriate DNA damage response mechanisms. The mammalian Protection Of Telomeres (POT1) protein interacts with single-stranded telomeric DNA and can exert positive and negative effects on telomere length. Of four distinct POT1 homologs in the roundworm Caenorhabditis elegans, deficiency for POT-1 or POT-2 resulted in progressive telomere elongation that occurred because both proteins negatively regulate telomerase. We created a POT-1::mCherry fusion protein that forms discrete foci at C. elegans telomeres, independent of POT-2, allowing for live analysis of telomere dynamics. Transgenic pot-1::mCherry repressed telomerase in pot-1 mutants. Animals deficient for pot-1, but not pot-2, displayed mildly enhanced telomere erosion rates in the absence of the telomerase reverse transcriptase, trt-1. However, trt-1; pot-1 double mutants exhibited delayed senescence in comparison to trt-1 animals, and senescence was further delayed in trt-1; pot-2; pot-1 triple mutants, some of which survived robustly in the absence of telomerase. Our results indicate that POT-1 and POT-2 play independent roles in suppressing a telomerase-independent telomere maintenance pathway but may function together to repress telomerase

Genesis of end-to-end chromosome fusions

Telomeres are DNA-protein complexes that form a protective cap at chromosome ends and provide a buffer against gradual loss DNA that occurs with every round of DNA replication. Telomere length is replenished by telomerase. Deficiency of telomerase in most human somatic cells causes telomere shortening with age. When telomeres become critically short, they uncap and can be processed as abnormal double strand breaks to generate end-to-end chromosome fusions. The resulting dicentric chromosomes may promote tumorigenesis, but they enter fusion-breakage-bridge cycles that impede the elucidation of the structure of the initial fusion event and a mechanistic understanding of their genesis. Current models for fusion of critically shortened, uncapped telomeres rely on PCR-based assays that typically capture fusion breakpoints created by ligation of two chromosome ends. We used two independent approaches that rely on distinctive features of the nematode C. elegans to study the frequency of direct end-to-end chromosome fusions in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusion events, and (2) unique subtelomeric sequences that allow for an unbiased, nearly exhaustive PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of initial end-to-end fusion events resulted from direct end-joining with no other rearrangements. We used three approaches to investigate complex fusion breakpoint structures: (1) physical analysis of the fusion breakpoint DNA by Southern blotting, (2) measurement of DNA copy number by microarray analysis, and (3) sequence analysis of fusion breakpoints recovered by inverse PCR. Duplications as large as two megabases were present at complex fusion breakpoints. Such events would have been missed by studies using typical PCR-based assays. Thus, duplications of various segments of the genome may be a major factor that drives end-to-end chromosome fusion and promotes tumor development

End Joining at Caenorhabditis elegans Telomeres

Critically shortened telomeres can be subjected to DNA repair events that generate end-to-end chromosome fusions. The resulting dicentric chromosomes can enter breakage–fusion–bridge cycles, thereby impeding elucidation of the structures of the initial fusion events and a mechanistic understanding of their genesis. Current models for the molecular basis of fusion of critically shortened, uncapped telomeres rely on PCR assays that typically capture fusion breakpoints created by direct ligation of chromosome ends. Here we use independent approaches that rely on distinctive features of Caenorhabditis elegans to study the frequency of direct end-to-end chromosome fusion in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusions and (2) unique subtelomeric sequences that allow for thorough PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of end-to-end fusion events resulted from direct end joining with no additional genome rearrangements. We also demonstrate that deficiency for the C. elegans Ku DNA repair heterodimer does not affect telomere length or cause synthetic effects in the absence of telomerase