Single molecule detection of direct, homologous, DNA/DNA pairing

doi:10.1073/pnas.0911214106

Single molecule detection of direct, homologous, DNA/DNA pairing

^aDepartment of Physics and
^bDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Contributed by Nancy Kleckner, September 30, 2009 (received for review August 10, 2009)

Abstract

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the “default” option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Footnotes

¹To whom correspondence may be addressed. E-mail: prentiss{at}fas.harvard.edu or kleckner{at}fas.harvard.edu
Author contributions: C.D., C.H.L., K.K., V.W.C., N.K., and M.P. designed research; C.D. and C.H.L. performed research; K.K. and K.H. contributed new reagents/analytic tools; C.D. and M.P. analyzed data; and C.D., N.K., and M.P. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0911214106/DCSupplemental.
↵* (i) If all moleules were present in homologous pairs, half of all pairs would contain either two biotin-labeled molecules or two Dig-labeled molecules and would not be detected. (ii) Each tethered bead represents two λDNAs rather than a single DNA as in the control sample.