Single molecule fret study on the mechanism of RecA mediated strand exchange

RecA plays a critical role during double strand break repair via homologous recombination. During the strand exchange reaction, RecA forms a helical filament on single stranded (ss) DNA that searches for homology and exchanges complementary base pairs with a homologous double strand (ds) DNA to form a new heteroduplex. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process which masks the subsequent strand exchange reaction. We developed a single molecule fluorescence assay with a few basepair and milliseconds resolution which can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggests that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Our model for strand exchange links structural models of RecA to its catalytic function. Next, we investigated the mechanism of RecA mediated homology search. Using tools with high spatiotemporal resolution to observe the encounter complex between the RecA filament and dsDNA, we present evidence in support of the “sliding model” wherein a RecA filament diffuses on a dsDNA track. Our results suggest that the sliding of the dsDNA relative to the RecA filament can explain the rapid changes in FRET which we have observed upon the docking of non-homologous dsDNA to the RecA filament. We further show that homology can be identified during such sliding. Sliding is thermally driven and occurs in the absence of ATP hydrolysis. Furthermore, homology recognition and basepairing can involve as few as 6 bp of complementarity. Our observation presents an example of how a multi-protein complex bound to DNA can serve as a vehicle enabling homology search processes via 1-D sliding. Finally, we demonstrate how an extension of the two color FRET assay to measure four colors simultaneously allows us to measure the correlation of reaction completion between the two ends of a single synaptic complex. We expect that this method will enable a multi dimensional analysis of independent reaction coordinates with broad applications in measuring the correlated dynamics of more complex biological systems

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Computnl Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2012

Author dc:creator

• Madabhusi Ragunathan, Kaushik

Contributors dc:contributor

• Ha, Taekjip
• Nair, Satish K.
• Spies, Maria
• Chemla, Yann R.

• DNA repair
• recombination
• single molecule
• Fluorescence resonance energy transfer (FRET)
• fluorescence
• Deoxyribonucleic acid (DNA)

Statement dc:rights

• Copyright 2012 Kaushik Madabhusi Ragunathan

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/32044

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/32044