POLQ- and CtIP-regulated telomere fusions and translocations are involved in early events in carcinogenesis

点击翻译按钮获取中文摘要

PROJECT SUMMARY We propose to investigate the mechanisms that regulate a cell’s ability to escape from the crisis caused by telomere shortening. As normal human cells age, their telomeres gradually shorten. When the telomeres shorten significantly, the cell undergoes senescence, which is a naturally occurring barrier to cancer. If, however, a cell should suffer a transforming mutation, it can by-pass senescence and continue to proliferate until its telomeres become so short that they are non-functional. The resulting lack of end protection triggers “crisis”, a state that is highlighted by genomic instability as chromosomes engage in breakage:fusion:bridging cycles that almost invariably result in the death of the cell. On rare occasions a cell can resolve its fusions, reestablish its telomeres and stabilize its genome. Such cells are said to be immortalized and it is likely that they are the progenitors of most human cancers. That the (dys)regulation of telomere maintenance is also associated with aging, immortalization, and tumorigenesis in other experimental systems adds confidence to the belief that these issues are conserved and important. Previously, we have demonstrated that DNA ligase III and poly (ADP) ribose polymerase 1 are required for human cells to survive crisis. Here, we propose to define the role of DNA polymerase theta/Q (POLQ), which acts in the same pathway, in this process. Unexpectedly, we show that deletion of POLQ causes telomere elongation and escape from crisis. We will uncover how POLQ normally suppresses these events. Integral to surviving crisis is a requirement to resolve the chromosomal fusions/translocations that occurred during crisis. One way to do this is to physically shear them apart by the application of tension (aka, “breakage”). This process, however, is highly mutagenic and often leads to lethal outcomes. A second resolution process more likely to ensure survival is to convert the chromosome fusions into ultra-fine bridges (UFBs) and then enzymatically — in a process that is very poorly understood — resolve these UFBs. Here, we demonstrate that the loss-of-function of the resection nuclease, C-terminal interacting protein (CtIP), results in a high frequency of UFBs that are not resolved and we propose experiments to mechanistically unravel how these bridges, many of which involve telomeres, are generated and why they are not resolved. In all of these approaches we utilize the strengths of the Hendrickson and Baird laboratories. The Hendrickson laboratory excels at the technology of gene targeting to study the impact of loss-of-function mutations of genes (POLQ and CtIP in this instance) on telomere maintenance. The Baird laboratory is the world’s leader in analyzing telomere fusion events in human cells undergoing crisis. Their ability to characterize the dynamics of single telomeric ends has provided the field’s deepest understanding of the mechanism of telomere fusions in human cells. In summary, our proposed studies impact on DNA repair and telomere maintenance and the importance of understanding these processes for cancer biology is clear.