Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Tuesday Speaker Abstracts

Single-Molecule Investigations of DNA Replication and Repair in Living Bacteria Julie Biteen . University of Michigan, Ann Arbor, MI, USA. By beating the diffraction limit that restricts traditional light microscopy, single-molecule fluorescence imaging is a precise, non-invasive way to sensitively probe position and dynamics. Our lab has been developing new methods to locate, track, and analyze single molecules to measure structure, dynamics, and cooperativity in live bacterial cells. I will discuss how we are measuring and understanding the dynamical interactions essential for DNA mismatch recognition and DNA replication in living Bacillus subtilis cells. In particular, we have integrated our nanoscopic biophysical tools with biochemical and genomic approaches to understand how the mismatch repair protein MutS efficiently identifies DNA mismatches during real time in living cells, to determine that the replicative polymerase PolC is continuously recruited to and released from a centrally located replisome, and to measure how the various components of the multi-protein replication machinery are organized in bacterial cells. Overall, our results provide a mechanistic model for the dynamical nature of these fundamental processes in live cells.

Single-Molecule Visualization of Bacterial DNA Repair in Live Cells Antoine M. van Oijen , University of Wollongong, Wollongong, NSW, Australia.

We are using single-molecule fluorescence methods to visualize the behaviour of individual, fluorescently labelled molecules inside living cells. Using these approaches, we aim to understand the relationship between DNA replication and repair. In particular, we study aspects related to the bacterial SOS damage response: how does the RecA protein coat single-stranded DNA after DNA damage stalls replication and how do the subsequently upregulated translesion synthesis DNA polymerases gain access to the lesions in the DNA? Further, we investigate the dynamics of transcription-coupled repair as mediated by the E. coli Mfd protein. These studies not only lead to new insights in molecular pathways, they also provide information on the critical role played by spatial and temporal regulatory mechanisms.

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