DNA double-strand break (DSB) repair is spatially organized into nuclear repair domains that specifically facilitate DSB repair by homologous recombination (HR). HR, one of the major DSB pathways along with non-homologous end-joining, has been implicated in tumorigenesis, notably following mutations in the tumor suppressor genes BRCA1 and BRCA2 [1, 2]. Our lab demonstrated that upon DSB formation by induction of a restriction endonuclease (RE) or treatment with neocarzinostatin (NCS), WASP activates ARP2/3, which polymerizes nuclear actin into branched filaments [4]. This enhances the mobility of DSBs destined for HR and their subsequent clustering into HR domains. The DNA topoisomerase II (Top2) inhibitor etoposide (ETO) yields DSBs harboring protein-DNA adducts that require resection and subsequent repair by HR factors, including MRN, CtIP, and BRCA1 [5, 6]. Because of the absolute requirement for poisoned Top2 removal prior to repair, ETO is a unique way to probe the functional relationship between resection and movement. ETO is used to treat a wide range of cancers, including leukemia and soft tissue cancers. However, treatment is associated with secondary leukemias due to translocations. Using live-cell imaging, I show that ETO DSBs undergo ARP2/3-mediated movement and clustering. However, unlike RE and NCS DSBs, movement is not restricted to G2 but also occurs in G1. Additionally, ETO breaks in G1 undergo resection and load HR machinery, such as RPA. I have also begun examining the role of HR factors, including Mre11 and BRCA2, in repair domain formation following the generation of DSBs by RE, NCS and ETO. Although DSB clustering is crucial for HR, little is known about how repair domains are formed and their local and genome-wide implications. For example, we do not fully understand the crosstalk between movement (actin, WASP) and repair (HR machinery) in mammalian cells. Additionally, the dynamics of DSBs likely influences chromosomal rearrangements. Our lab is integrating high-throughput genomic technologies that assess gene- gene interactions and translocation events to determine the genome-wide implications of DSB mobility. The overarching goals of this study are to elucidate mechanisms by which nuclear actin polymerization and HR proteins regulate repair domain formation and to evaluate the genome-wide impact of DSB mobility. I hypothesize that HR proteins, including the resection machinery, play a critical role in regulating ARP2/3- mediated DSB movements and subsequent clustering. I further propose that nuclear actin polymerization impacts genome organization following DNA damage and thus affects translocation frequency. I will investigate these hypotheses in the following aims:
Aim 1 : Elucidate the contribution of HR machinery to Arp2/3-dependent DSB clustering.
Aim 2 : Determine the impact of ARP2/3-mediated DSB movement on genome stability.
This project seeks to understand the mechanisms of DNA double-strand break repair domain formation with special emphasis on the role of homologous recombination proteins. We will also elucidate the impact of DNA clustering on genome organization and integrity. Understanding the processes that undermine repair domain formation and yield pathological consequences may reveal novel chemotherapeutic targets or provide a way to prevent genotoxic effects of current chemotherapy regimens.
