Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

• Published in: Nature (London) 2012-02, Vol.482 (7385), p.423-427
• Main Authors: Forget, Anthony L., Kowalczykowski, Stephen C.
• Format: Article
• Language: English
• Subjects: 631/337/1427/2190; 631/45/535; 631/57/2265; Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Bacteriophage lambda - genetics; Base Pairing; Biological and medical sciences; Deoxyribonucleic acid; DNA; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA, Viral - chemistry; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Homologous Recombination; Humanities and Social Sciences; letter; Microscopy, Fluorescence; Molecular and cellular biology; multidisciplinary; Nucleic acids; Proteins; Real time; Rec A Recombinases - metabolism; Recombinational DNA Repair; Science; Science (multidisciplinary); Sequence Homology, Nucleic Acid; Sucrose
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature10782

The search for DNA homology is vital to recombinational DNA repair and occurs by intersegment contact sampling wherein the three-dimensional conformational state of the double-stranded DNA target and the length of the homologous RecA–single-stranded DNA filament have important roles. Reducing complexity to manageable size During DNA repair by homologous recombination, the molecule containing a double-strand break must find an undamaged, intact, exact copy of the sequence at the break to initiate a process known as strand exchange. This is facilitated by cooperative binding of a RecA family strand-exchange protein to a single-strand tail of the broken DNA. But how does the RecA–DNA filament find its matching sequence, which represents a tiny fraction of the total DNA content? In a new study, Anthony Forget and Stephen Kowalczykowski show that weak, transient contacts by the RecA–DNA filament with duplex DNA allow it to sample three-dimensional space to accelerate the recognition of homologous sequence. 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 1 . 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 2 , 3 , 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 p