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King Lab
DNA Repair
We (and others) have shown that persistent double-stranded DNA breaks (DSBs) associate with the nuclear periphery. We are currently investigating the signals and biochemical interactions that drive this association as well as the biological consequence of coupling DSBs to the LINC complex.
Damage
Chromatin and Nuclear Envelope Structure
In order to investigate how chromatin contributes to nuclear mechanics in a quantitative manner, we are establishing methods to apply force spectroscopy to isolated S. pombe nuclei through a collaboration with the laboratory of Simon Mochrie Ph.D. (Physics). We can manipulate isolated nuclei, associate them with a glass surface and bind beads to the nuclei. Using an optical trap, we can induce repeated rounds of compression and extension and extract quantitative information about nuclear elasticity. This assay is being applied to nuclei with different chromatin to nuclear volume ratios and perturbations in chromatin-nuclear envelope attachments.
tweezers
Interface of LINC Complexes and Microtubules
Although we know that LINC complexes bridge the nuclear envelope to connect chromatin and the cytoskeleton, the molecular players at the two interfaces remain undefined. We are using biochemical and genetic methods to identify proteins capable of coupling the integral inner nuclear membrane protein Sad1 to chromatin within the nucleus and the integral outer nuclear membrane KASH proteins, Kms1 and Kms2, to microtubules (MTs) in the cytoplasm. In addition, we are building a reconstituted system using isolated nuclei and MTs to probe the kinetics of LINC-MT interface formation.
interface

Cartoon of the unidentified bridging components we propose to identify.
A. Adapters between KASH proteins and microtubules
B. Additional membrane proteins
C. Adapters between Ima1 and chromatin

Dividing S. pombe with GFP labeled Cut11, spindle pole body docking protein.

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S. pombe during meiosis.
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Current Research is Funded by:
NIH Director's New Innovator Award
Searle Scholar 2011

Membership:
Yale Cancer Center
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