78
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.