Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology
search
Nature 482, 7385 (2012). doi:10.1038/nature10782
Authors: Anthony L. Forget & Stephen C. Kowalczykowski
DNA breaks can be repaired with high fidelity by homologous recombination. A
ubiquitous protein that is essential for this DNA template-directed repair is
RecA. After resection of broken DNA to produce single-stranded
DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity
to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous
to the ssDNA. This homology search is vital to recombinational DNA repair, and
results in homologous pairing and exchange of DNA strands. Homologous pairing
involves DNA sequence-specific target location by the RecA–ssDNA complex.
Despite decades of study, the mechanism of this enigmatic search process remains
unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA
pairing and strand exchange, eliminating active search processes.
Using dual optical trapping to manipulate DNA, and single-molecule fluorescence
microscopy to image DNA pairing, we demonstrate that both the three-dimensional
conformational state of the dsDNA target and the length of the homologous
RecA–ssDNA filament have important roles in the homology search. We
discovered that as the end-to-end distance of the target dsDNA molecule is
increased, constraining the available three-dimensional (3D) conformations of the
molecule, the rate of homologous pairing decreases. Conversely, when the length of
the ssDNA in the nucleoprotein filament is increased, homology is found faster. We
propose a model for the DNA homology search process termed ‘intersegmental
contact sampling’, in which the intrinsic multivalent nature of the RecA
nucleoprotein filament is used to search DNA sequence space within 3D domains of
DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings
highlight the importance of the 3D conformational dynamics of DNA, reveal a
previously unknown facet of the homology search, and provide insight into the
mechanism of DNA target location by this member of a universal family of
proteins.
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