Telomere maintenance pathways and their role in genome stability.

Most cancer cells have the capacity to replicate indefinitely. This is partly achieved by activation of a telomere maintenance mechanism (TMM) and we are trying to understand the molecular basis of some of these pathways. Telomeres are repetitive DNA sequences that cap chromosomes and so prevent damage to the genome. The majority of tumours activate telomerase or the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomeres but the TMM is unknown in a subset of tumours. In addition the molecular mechanism that underlies ALT is poorly understood. Recently we have shown that activation of the ALT pathway can destabilize other repeated sequences in the genome but the significance of this observation is unclear. Therefore using single telomere and single molecule approaches we plan to investigate telomeric DNA and other sequences in order to elucidate the underlying TMM in tumours. In addition in cell lines we plan to disrupt the expression of genes that are thought to play a role in ALT and study the effect on telomeres and other DNA sequences in the genome. In the long term we hope this will lead to identification of targets for novel anti-cancer therapies.

We have shown that human telomere repeat arrays are prone to mutations, with a germline mutation rate of 0.6% per kb. We proposed that the germline mutation process is dominated by intra-allelic events and that inter-allelic recombination is uncommon. Our recent analysis of sequences adjacent to the Xp/Yp telomere, encompassing a hyper variable minisatellite (DXYS14), strongly supports this view. We have also shown the telomere mutation frequency is significantly higher in sporadic colon cancers, increasing to 35% per allele in tumours with defects in mismatch repair (MMR). Again the mutation process appears to be dominated by intra-allelic events. In contrast complex telomere mutations, discovered in cells that use the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomere length, are highly likely to arise by recombination-like processes that underlie ALT. Control of the ALT pathway and genes required for telomere lengthening are poorly understood. Recently we have shown that the MS32 minisatellite, which normally mutates by recombination in the human germline, becomes highly unstable upon activation of the ALT pathway but four other minisatellites remain stable. To investigate the link between the minisatellite and telomere instability in ALT+ cell lines we will measure mutation frequencies at a variety of minisatellites, selected by their location in the genome and the nature of the repeat array (sequence composition, repeat length). To determine whether the destabilization of MS32 is a localized perhaps chromatin mediate effect, we will study other repeated sequences in the vicinity of the MS32 locus and we will examine gene expression in related cell lines with and without ALT activity. We will investigate the frequency of extraordinary MS32 instability in soft tissue and other sarcomas to determine whether it has value as diagnostic marker. We also plan to utilize the MS32 minisatellite as a tool to dissect the ALT pathway. Initially we will disrupt the expression of helicase genes thought to play a role in ALT and study the effect on the mutation rate and profile at the MS32 locus and at single telomeres. We plan to take a similar approach to study the effect of disruption of the MMR pathway on telomere stability and length. Finally we plan to develop PCR based tools for the analysis of a subset of telomeres in the mouse. Such tools will be valuable in the investigation of the ALT pathway and of telomere length dynamics in mutant strains that model some human diseases.