Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

49-POS Board 49 RecA E38K Mutant Displaces SSB without Apparent ssDNA Length Dependence Hung-Yi Wu 1 , Tzu-Ling Tseng 1 , Michael M. Cox 2 , Hung-Wen Li 1 . 1 National Taiwan University, Taipei, Taiwan, 2 University of Wisconsin-Madison, Madison, WI, USA. RecA recombinases catalyze the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments as the first step of the repair process. Previously, it has been suggested that RecA competes with SSB by binding and extending onto the free ssDNA region. However, the detailed displacement process is not clear. In this study, we monitored individual RecA filament formation by single-molecule tethered particle motion (TPM) technique in real-time. Binding of RecA on the SSB wrapped- ssDNA extends the DNA substrate, visible by the increase in bead Brownian motion in the TPM imaging. We found a point mutation on RecA (E38K) accelerates the SSB removal rate. By varying the length of ssDNA gap from 60 to 100 nucleotides (60, 65, 70, 72, 75, 78, 80, 90, 100), in which only one SSB binding is allowed using the 65mer wrapping mode, we found the nucleation time of RecA E38K to form extended nucleoprotein filament is roughly constant over the ssDNA gap size used. This and control experiments suggest that the length of ssDNA is not critical for the nucleation process, an observation different from the long-believed, expected passive nature of RecA nucleation.

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