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Single Molecule Imaging

David Rueda

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Medical Research Council (MRC)
The overall aim of the program is to develop single-molecule and super-resolution imaging methodologies to monitor key cellular processes in real time. In recent years, cellular imaging has witnessed a remarkable revolution with the advent of single molecule spectroscopy and super-resolution microscopy. These technologies enable the direct observation of biochemical pathways in real time, and to characterize them in unprecedented detail. Specifically, our group focuses on three main topics: RNA Splicing is an essential step in the maturation of eukaryotic pre-mRNAs. Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous diseases such as breast, colorectal, epithelial and ovarian cancers, as well as neurodegenerative disorders such as Parkinsons and Alzheimers. The spliceosome is a large dynamic assembly of 5 small nuclear RNAs (snRNA) and over 100 proteins that catalyzes splicing. It undergoes several, highly conserved, conformational rearrangements. The active site of the spliceosome comprises two key snRNAs (U2 and U6) that have been shown to undergo splicing-related catalysis in absence of proteins. The structure and dynamics of the U2-U6 complex are thought to play critical roles in the mechanism of splicing in vivo. Using active spliceosomes in yeast cell extracts reconstituted with flurophore-labeled U6 snRNA, we explore the role of these dynamics in splicing assembly and catalysis. The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3-hydroxyl of the growing DNA chain. However, the mechanism by which they achieve their extraordinary accuracy remains unclear. Specifically, kinetic intermediates involved in proofreading have never before been characterized. We use single-molecule approaches to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. The APOBEC family of enzymes comprises single-stranded ssDNA cytosine deaminases that play important roles in eliminating retroviral infectivity and somatic hypermutation (SHM). For example, APOBEC3G eliminates HIV1 infectivity by converting C→U in numerous small target motifs on the minus viral cDNA, wheras AID generates advantageous mutations in the variable region of immunoglobulin genes in B-cells that increase the affinity of antibodies for antigenes. Compared with dsDNA scanning enzymes (e.g., DNA glycosylases) that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. We investigate ssDNA scanning and motif-targeting mechanisms for Apo3G and AID using single molecule fluorescence. We address specific issues of deamination activity within the general context of ssDNA scanning mechanisms.

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