טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentOfir Rache
SubjectReplication of Human Telomeric Regions - Relevance to
Cellular Senescence
DepartmentDepartment of Medicine
Supervisor Professor Karl Skorecki


Abstract

Telomeres are the DNA and protein structures present at the ends of eukaryotic chromosomes. Their proper functioning is vital for maintenance of chromosomal integrity . In the vast majority of eukaryotes, telomeric DNA is composed of tandem arrays of short (5-26bp) repeat sequences. Human telomeres are composed of tandem repeats of the TTAGGG hexamer.  Passive telomeric DNA shortening occurs as an inevitable consequence of incomplete DNA replication of linear chromosome ends. Telomeres have been shown in yeast to induce late replication in S-phase and to silence transcription of neighboring genes.

In the current research we examined the replication timing of human telomeric regions and investigated the effect of proximity to telomere, cellular ageing and telomere length on the replication timing of genomic regions adjacent to human telomeres. The effect of proximity to telomere on adjacent regions in human chromosomes, was studied on cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats. Using fluorescent in-situ hybridization (FISH) of S-phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22. The difference in replication timing between the two chromosomes was not associated with apparent differences in the expression properties of ARSA, a gene located at a distance of approximately 54 kb from the breakpoint. We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect, which delays the replication of adjacent chromosomal regions in humans.


We next aimed to determine the replication timing of human telomeric regions and the effect of cellular ageing on replication timing of these regions. We have conducted the FISH replication timing assay in primary human foreskin fibroblasts utilizing cosmid probes which hybridize to several different telomeric regions in comparison to non-telomeric regions. In early passage cells, seven telomeric regions which we examined replicated during middle to late in S-phase. We proceeded to explore the relationship between changes in telomere replication and advanced cell passage and found a delay in the replication of a number of telomeric regions in pre-senescent human fibroblasts. Furthermore, we find a significant percentage of telomeric regions in metaphase chromosomes that are not replicated in pre-senescent cells. This phenomenon was reversed by prolonging metaphase. Progression in the cell cycle despite incomplete replication of telomeric regions would be expected to result in breakage of chromosome ends and formation of dicentric chromosomes as observed in senescent cells .