The Impact of Nucleases on Telomere Maintenance and Ultrafine Bridge Resolution

Abstract

Nucleolytic processing represents a pivotal step in repair of DNA double-stranded breaks, rescue of collapsed or stalled replication forks, and the maintenance of an error-free genome. Moreover, the ends of chromosomes, known as telomeres, frequently require nuclease action as they are difficult to replicate regions, which are prone to fusion. Further, mutation of known telomere-associated proteins leads to rampant telomere attrition and cellular death. To explore the putative function of a specific nuclease – Exonuclease 1; EXO1 – at telomeres, I generated EXO1 knockout cell lines in 4 distinct genetic backgrounds. My research showed that EXO1 is specialized in facilitating replication through G-quadruplex secondary structures in the telomere. Resection proximal to these structures is essential for complete replication of telomeres and the absence of EXO1 renders telomeres both shorter and enhances their dysfunction. Finally, I explored EXO1 inhibition and G-quadruplex stabilization as a plausible coupled cancer therapeutic approach. I also sought to explore the function(s) of the C-terminal Interacting Protein (CtIP) endonuclease in mammalian telomere maintenance. Importantly, I was able to documente a bona fide role for CtIP in generating the overhang which exists at mammalian telomeres. Additionally, I demonstrated that replication of the entire telomere is compromised in the absence of CtIP, leading to elevated dysfunction and telomeric loss. Most striking of all was the accumulation of t-circles, which consist of telomeric DNA that has been liberated from chromosomes, presumably due to failed replication. Altogether, telomeres shortened rapidly if CtIP was not present to participate in both the overhang generation and replication maintenance pathways. Finally, I observed prominent anaphase abnormalities upon CtIP removal. This led me to explore CtIP’s role in mitosis and in the processing of both chromatin and ultrafine bridges. I showed that the nuclease function of CtIP along with the Bloom Syndrome (BLM) helicase is essential for converting double-stranded ultrafine bridges into single-stranded ultrafine bridges. This conversion prevents the accumulation of single-stranded ultrafine bridges in late anaphase, which, in turn, protects the genome from the frank loss of DNA and the accumulation of DNA damage. Altogether, I have documented novel roles for the CtIP and EXO1 nucleases in telomere maintenance and anaphase bridge processing.