Telomere Specific Homologous Recombination in the Alternative Lengthening of Telomeres

Over twenty years have passed since the discovery of telomerase-independent telomere maintenance, yet the precise details of the ALT mechanism remain a mystery. A growing body of evidence suggests that ALT cells maintain telomeres by homologous recombination (Reddel 2003 for Review). Groundbreaking work by Oliver Bechter demonstrated that ALT cells and telomerase-positive cells show no difference in the rate of general HR. This study fundamentally shaped our current concept of the ALT mechanism, implying that it involves preferential recombination of telomeric repeats. Since ALT seems to require proteins involved in normal HR, it follows that this telomeric recombination must be suppressed in telomerase positive or normal cells. However, to date there has been no direct evidence to support this hypothesis. Seeking to investigate the rates of telomere specific recombination, previous work in the Shay and Wright lab utilized the Tel-Tel vector. However, due to the method of integration only a limited number of clones could be analyzed and no statistically significant conclusions could be made. My work has focused on remedying this limitation. I have developed a strategy for integrating the Tel-Tel vector into a variety of host cell lines and generating a large number of distinct clones for each line. Using this strategy I was able to measure the average rates of telomeric HR for each cell line and provide the first direct evidence that ALT cells show distinctly elevated levels of increased telomere-specific HR. Additionally, I have constructed a control vector which functions in the same manner as Tel-Tel, differing only in that the telomeric repeats are replaced by non-telomeric repeat sequence. Using this vector (referred to as Mut-Mut) and the same incorporation method, I have demonstrated that there is no significant difference in the rates of general HR in ALT and telomerase positive cells. Finally, I used this novel ALT reporter as well as previously established methods to investigate some proteins that may play a central role in the ALT mechanism.