Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology             search.
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.
Department of Microbiology, University of California, Davis, California 95616-8665, USA.
Anthony L
AL
Forget
Stephen C
SC
Kowalczykowski
D000255:Adenosine Triphosphate / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D020029:Base Pairing; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D059765:Homologous Recombination; D008856:Microscopy, Fluorescence; D011938:Rec A Recombinases / Q000378:metabolism*; D059767:Recombinational DNA Repair; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases