DNA double-strand break chromatin alterations and genome integrity

University Of Pennsylvania, Philadelphia PA

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Abstract

Acquisition of a fluorescent microscope for genetic interrogation of DNA damage responses Project Summary/Abstract: Alternative lengthening of telomeres (ALT) is poorly understood homology directed repair mechanism that is responsible for telomere maintenance in ~15% of human cancers. We have developed systems that enable quantitative, real-time visualization of each step during ALT. Our published studies from this R01 reveal that ALT is initiated by DNA damage dependent clustering of telomeres into nuclear bodies that serve as hubs for long tract homology directed repair synthesis. We named this mechanism Break Induced Telomere Synthesis and showed that it is critical for telomere lengthening in cells that utilize ALT. To examine the question of how DNA damage responses assemble on telomeric chromatin, we coupled synchronous telomere DNA double-strand break induction with telomere purification to identify the telomere DNA damage response proteome. This revealed a hybrid damage response that consists of both homology directed DNA repair and replication stress factors. We hypothesize that ALT occurs by assembling elements of several different DNA repair mechanisms on telomeric chromatin to achieve long tract homology directed repair synthesis. Our preliminary data reveals that responses that either promote or inhibit ALT. To systematically address this response, it is essential for us to perform loss of function experiments that allow us to quantitatively assess the importance of each factor during ALT. The overall goal of this proposal is to perform arrayed CRISPR screens that target each factor in the telomere damage response. We will visualize the effect on ALT by quantifying EdU colocalization at telomeres in G2 as a marker of recombination dependent telomere maintenance. This is a known hallmark of ALT. We will use the Cas9 system that allows guide RNAs targeted to functional domains in each gene. The screen will be performed in unperturbed ALT cells and following TRF1-FokI induction, which is a telomere specific nuclease that generates DNA double-strand breaks at telomeres that promote ALT. Imaging will be performed in an arrayed format on a Nikon Ti2E motorized inverted microscope that is equipped with the capacity to image either live or fixed cells with a 40X objective in a 96 well format. These fundamental studies will allow us to define the critical elements of the telomere DNA damage response and how it allows telomere lengthening for sustained proliferation in cells that rely on ALT.