Double-strand breaks (DSBs) are DNA lesions that yield loss of genetic material when unrepaired and chromosome rearrangements when mis-repaired. Abrogating the repair of DSBs generated by chemotherapy and ionizing radiation remains a major goal in the development of more effective cancer treatments. In mammalian cells and yeast, DSBs migrate into clusters, which is postulated to concentrate repair factors in centers to favor more efficient repair7,8,16. In particular, homology-directed repair (HDR) is associated with increased chromosomal mobility and clustering5,6,9,. The molecular basis for DSB movement however, remains enigmatic. The machinery that drives actin polymerization is found in the nucleus23,30,31,32. Notably, the Arp2/3 complex, an actin nucleation promoting factor, as well as its activator WASP, a Wiskott-Aldrich syndrome family member, are known to generate propulsive forces by nucleating a highly-branched network of actin filaments15. Recently, genotoxic agents were shown to trigger actin polymerization in the nucleoplasm of mammalian cells11. However, the role of actin filaments in DSB repair is not characterized. Recently, we performed a proteomic screen using liquid chromatography mass spectrometry to identify novel proteins recruited to damaged chromatin in Xenopus laevis cell-free extracts. Surprisingly, we observe enrichment of the Arp2/3 complex, ?-actin, and actin filament capping proteins in DSB-containing chromatin relative to undamaged controls. In mammalian cells, we find that the Arp2/3 complex co-localizes with WASP at DSBs destined for repair by HDR. Following genome-wide generation of DSBs, we observe clustering of DSB foci, particularly in S and G2 when HDR predominates. Notably, we show that inhibition of actin polymerization by small molecule inactivation of WASP or Arp2/3 reduces DSB clustering and repair by homology-directed mechanisms. We propose, therefore, that the Arp2/3 complex is poised to play a critical role in the polymerization of actin polymers that cluster DSBs for HDR. The overarching goal of this study is to understand the role actin polymerization plays in DSB mobility and investigate whether Arp2/3 inhibitors synergize with DNA damaging chemotherapies. Given that WASP and the Arp2/3 complex are enriched in damaged chromatin, I hypothesize that nuclear actin polymerization drives DSB clustering for DSB repair. I will investigate this hypothesis in the following three aims:
Aim 1 : Characterize the spatiotemporal dynamics of nuclear actin polymerization during DSB repair Aim 2: Investigate actin-driven chromosomal motion during DSB repair and translocation formation Aim 3: Assess whether Arp2/3 inhibitors chemosensitize tumor cells to DNA damaging therapies
This project seeks to understand the proteins responsible for chromosomal mobility in the nucleus and how their inhibition may compromise DNA repair. Uncovering new processes that undermine DNA repair may reveal novel ways to optimize genotoxic forms of chemotherapy.
| Schrank, Benjamin R; Aparicio, Tomas; Li, Yinyin et al. (2018) Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 559:61-66 |
