Genesis of end-to-end chromosome fusions

Abstract

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