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dna av1

Dissecting the regulation of DNA replication and repair using imaging, conditional genetics and cell biology. A. Phosphorylation of the heterochromatin protein HP1 at sites of DNA damage (see Ayoub et al Nature (2008)). B. Transient mobilization of HP1 followed by accumulation at sites of DNA damage controls early events in DNA damage signaling (see Venkitaraman et al Crit Rev Biochem Mol Biol (2010)). C. Exploiting the N-end rule to construct a thermosensitive protein ‘degron’ as a general tool for conditional genetics in vertebrate cells. The right-hand panel shows the depletion and restitution of an an EGFP-degron with temperature shifts (see Su et al Nature Str Mol Biol (2008) and Bernal et al J Cell Biol (2011)).

imaging

Probing cellular function and tissue architecture using new methods in imaging. CLICK HERE FOR A HIGH RESOLUTION IMAGE. A. Combining somatic cell genetics with fluorescence correlation spectroscopy for ‘in vivo biochemistry’ – molecular complexes formed by BRCA2 and their alteration after DNA damage (see Jeyasekharan et al Proc Nat Acad Sci USA(2010)). B. New FRET sensors for imaging biochemical reactions in DNA damage response pathways. C. Combining somatic cell genetics with fluorescence correlation spectroscopy for ‘in vivo biochemistry’ – reaction-diffusion mechanisms corral PLK1 kinase in the centrosome (see Mahen et al Proc Nat Acad Sci USA (2011)). D. Second harmonic generation (SHG) imaging microscopy, hyperdimensional imaging microscopy (HDIM) and H&E staining of histological sections. SHG and HDIM images are acquired with a non-labeled sample but provide high-contrast images.

chro av2

Analyses of mitotic progression and the completion of cell division using RNAi screening, biochemistry and imaging. CLICK HERE FOR A HIGH RESOLUTION IMAGE. A. High-throughput, high-content RNAi screening identifies UBE2S as a regulator of mitotic exit after taxol-induced mitotic arrest. B. The function of UBE2S as an ‘E4’ cofactor for the APC/C E3 ligase is revealed by biochemistry C. A two-step model for human APC/C function in substrate degradation leading to mitotic exit (see Garnett et al Nature Cell Biol (2009)). D. A role for Orc6 in cytokinesis revealed by combining a temperature sensitive degron-Orc6 with fluorescence photobleaching (see Bernal & Venkitaraman J Cell Biol(2011)).

CBMT image

Interdisciplinary approaches to extending the repertoire of ‘druggable’ targets. CLICK HERE FOR A HIGH RESOLUTION IMAGE. (a) A two dimensional representation of chemical space being partitioned for library construction and hit expansion into clusters of similar compounds using a simple sphere exclusion method (see Huggins et al, ACS Chemical Biology (2011). (b) Guiding the rational synthesis of chemical inhibitors against novel target classes. The panels show the X-ray crystal structures of a protein-protein interaction domain with and without a bound small-molecule inhibitor. (c) Probing biological function with small molecule inhibitors. The normal metaphase alignment of chromosomes (top row) is contrasted against the effects (bottom row) of an inhibitor of a mitotic protein-protein interaction.

brca2 av4

Analysis of the pathogenesis and therapy of BRCA2-mutant cancers using genetics and molecular cell biology. A. Chromosomal instability evoked by BRCA2 inactivation provides the first insights into its role in mitotic recombination (see Patel et al Mol Cell (1998)). B. Aneuploidy in BRCA2-deficient cells associated with defects in the completion of mitotic cell division (see Daniels et al Science (2004), Lee et al Oncogene (2011)). C. Progression of pancreatic neoplasia (top panel) and therapeutic responsiveness to Olaparib (bottom panel) revealed in a model for tissue-specific carcinogenesis associated with BRCA2 inactivation summarized in D (see Skoulidis et al Cancer Cell (2010)).

brac2 av3

A. Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombination enzyme (see Rajendra & Venkitaraman, Nuc Acids Res (2010)). B. A schematic illustration summarizing the functions of BRCA2 in the assembly of RAD51 filaments on damaged DNA (see Venkitaraman, Ann Rev Pathol (2009)). C. BRC repeats delay dsDNA capture (top panel) coincident with joint molecule formation (bottom panel) in a reconstituted RAD51-mediated DNA recombination reaction (see Shivji et al Proc Nat Acad Sci USA (2009)). D. Opposing actions of the BRC repeats stabilize ssDNA-RAD51 binding whilst inhibiting dsDNA-RAD51 capture to coordinate RAD51-mediated strand exchange (see Shivji et al Proc Nat Acad Sci USA (2009)).

DNArep.png

CLICK HERE FOR A HIGH RESOLUTION IMAGE. A. Phosphorylation of the heterochromatin protein HP1 at sites of DNA damage (see Ayoub et al Nature (2008)). B. Transient mobilization of HP1 followed by accumulation at sites of DNA damage controls early events in DNA damage signaling (see Venkitaraman et al Crit Rev Biochem Mol Biol (2010)). C. Exploiting the N-end rule to construct a thermosensitive protein ‘degron’ as a general tool for conditional genetics in vertebrate cells. The right-hand panel shows the depletion and restitution of an an EGFP-degron with temperature shifts (see Su et al Nature Str Mol Biol (2008) and Bernal et al J Cell Biol (2011)).

Chrseg.png

CLICK HERE FOR A HIGH RESOLUTION IMAGE. A. High-throughput, high-content RNAi screening identifies UBE2S as a regulator of mitotic exit after taxol-induced mitotic arrest. B. The function of UBE2S as an ‘E4’ cofactor for the APC/C E3 ligase is revealed by biochemistry C. A two-step model for human APC/C function in substrate degradation leading to mitotic exit (see Garnett et al Nature Cell Biol (2009)). D. A role for Orc6 in cytokinesis revealed by combining a temperature sensitive degron-Orc6 with fluorescence photobleaching (see Bernal & Venkitaraman J Cell Biol (2011)).

MolImaging.png

CLICK HERE FOR A HIGH RESOLUTION IMAGE. A. Combining somatic cell genetics with fluorescence correlation spectroscopy for ‘in vivo biochemistry’ – molecular complexes formed by BRCA2 and their alteration after DNA damage (see Jeyasekharan et al Proc Nat Acad Sci USA(2010)). B. New FRET sensors for imaging biochemical reactions in DNA damage response pathways. C. Combining somatic cell genetics with fluorescence correlation spectroscopy for ‘in vivo biochemistry’ – reaction-diffusion mechanisms corral PLK1 kinase in the centrosome (see Mahen et al Proc Nat Acad Sci USA (2011)). D. Second harmonic generation (SHG) imaging microscopy, hyperdimensional imaging microscopy (HDIM) and H&E staining of histological sections. SHG and HDIM images are acquired with a non-labeled sample but provide high-contrast images.

CBMT_image.png

CLICK HERE FOR A HIGH RESOLUTION IMAGE. (a) A two dimensional representation of chemical space being partitioned for library construction and hit expansion into clusters of similar compounds using a simple sphere exclusion method (see Huggins et al, ACS Chemical Biology (2011). (b) Guiding the rational synthesis of chemical inhibitors against novel target classes. The panels show the X-ray crystal structures of a protein-protein interaction domain with and without a bound small-molecule inhibitor. (c) Probing biological function with small molecule inhibitors. The normal metaphase alignment of chromosomes (top row) is contrasted against the effects (bottom row) of an inhibitor of a mitotic protein-protein interaction.

vent.jpg

Analysis of the pathogenesis and therapy of BRCA2-mutant cancers using genetics and molecular cell biology. A. Chromosomal instability evoked by BRCA2 inactivation provides the first insights into its role in mitotic recombination (see Patel et al Mol Cell (1998)). B. Aneuploidy in BRCA2-deficient cells associated with defects in the completion of mitotic cell division (see Daniels et al Science (2004), Lee et al Oncogene (2011)). C. Progression of pancreatic neoplasia (top panel) and therapeutic responsiveness to Olaparib (bottom panel) revealed in a model for tissue-specific carcinogenesis associated with BRCA2 inactivation summarized in D (see Skoulidis et al Cancer Cell (2010)).

BRCA2_Image2.jpg

[Click image for a high resolution image] A. Chromosomal instability evoked by BRCA2 inactivation provides the first insights into its role in mitotic recombination (see Patel et al Mol Cell (1998)). B. Aneuploidy in BRCA2-deficient cells associated with defects in the completion of mitotic cell division (see Daniels et al Science (2004), Lee et al Oncogene (2011)). C. Progression of pancreatic neoplasia (top panel) and therapeutic responsiveness to Olaparib (bottom panel) revealed in a model for tissue-specific carcinogenesis associated with BRCA2 inactivation summarized in D (see Skoulidis et al Cancer Cell (2010)).