DNA double-strand break chromatin alterations and genome integrity

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Contact PD/PI: Greenberg, Roger A. Summary Evolutionary conserved DNA repair mechanisms cooperate throughout the cell cycle to faithfully maintain genome integrity. However, a minimal understanding exists for how damaged chromatin directs the series of events that dictate ensuing repair mechanism. This gap in knowledge is particularly relevant to a poorly understood homology directed repair mechanism that becomes active to maintain telomere length in approximately 15% of human cancers. Alternative lengthening of telomeres (ALT) occurs by noncanonical homology directed DNA repair mechanism in cancers of mesenchymal origin. The recombination mechanisms responsible for telomere maintenance represent a unique vulnerability in ALT reliant cancers. We have developed experimental systems that enable quantitative, real-time visualization of each step in the homology directed repair of damaged telomeres. Our published studies reveal that ALT is initiated by DNA damage dependent homology directed repair synthesis that proceeds unidirectionally to the end of the chromosome. We named this mechanism Break Induced Telomere Synthesis and showed that it is critical for telomere lengthening in cells that utilize ALT. Here, we examine the question of how DNA damage responses assemble on telomeric chromatin to direct long-tract homology directed repair telomere synthesis. We hypothesize that Break Induced Telomere Synthesis occurs by assembling elements of several different DNA repair mechanisms on telomeric chromatin to achieve homology directed repair synthesis. Our unpublished results definitively support this premise, showing extensive PCNA- ubiquitination and recruitment of the 5’-3’ nuclease SNM1A and 5’-3’ helicase FANCJ at damaged telomeres to promote DNA repair synthesis and telomere integrity. We will investigate the basis for their damage dependent association with telomeres and functions in recombination dependent repair synthesis at telomeres using a multifaceted approach that allows us to examine each step of the telomere damage response in cells and in vitro in highly purified systems that assemble the break induced replisome on defined substrates. These fundamental studies are designed to understand the critical elements of Break Induced Telomere Synthesis and how it allows telomere lengthening for sustained proliferation in cells that rely on ALT.