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Prentiss, Mara

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 atDNA 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.

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3'3' ends differs from that which results when it is pulled from the 5'5' ends. In this work, we demonstrate, using lambda phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3'3' pulling versus 5'5' pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 +/- 3 pN and 122 +/- 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 +/- 3 pN for 3'3' and 110 +/- 3 pN for 5'5'; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 +/- 0.8 pN microm for 3'3' versus 13 +/- 8 pN for 5'5'; and (iv) 3'3' and 5'5' molecules show different changes in hysteresis due to interactions with beta-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.