RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA-RecA filaments.

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.

Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA.

RecA is a key protein in homologous recombination. During recombination, one single-stranded DNA (ssDNA) bound to site I in RecA exchanges Watson-Crick pairing with a sequence-matched ssDNA that was part of a double-stranded DNA molecule (dsDNA) bound to site II in RecA. After strand exchange, heteroduplex dsDNA is bound to site I. In vivo, direct polymerization of RecA on dsDNA through site I does not occur, though it does in vitro. The mechanisms underlying the difference have been unclear. We use single-molecule experiments to decouple the two steps involved in polymerization: nucleation and elongation. We find that elongation is governed by a fundamental clock that is insensitive to force and RecA concentration from 0.2 and 6 µM, though rates depend on ionic conditions. Thus, we can probe nucleation site stability by creating nucleation sites at high force and then measuring elongation as a function of applied force. We find that in the presence of ATP hydrolysis a minimum force is required for polymerization. The minimum force decreases with increasing RecA or ATP concentrations. We propose that force reduces the off-rate for nucleation site binding and that nucleation site stability is the stringency factor that prevents in vivo polymerization.

Study of force induced melting of dsDNA as a function of length and conformation.

We measure the constant force required to melt double-stranded (ds) DNA as a function of length for lengths from 12 to 100,000 base pairs, where the force is applied to the 3'3' or 5'5' ends of the dsDNA. Molecules with 32 base pairs or fewer melt before overstretching. For these short molecules, the melting force is independent of the ends to which the force is applied and the shear force as a function of length is well described by de Gennes theory with a de Gennes length of less than 10 bp. Molecules with lengths of 500 base pairs or more overstretch before melting. For these long molecules, the melting force depends on the ends to which the force is applied. The melting force as a function of length increases even when the length exceeds 1000 bp, where the length dependence is inconsistent with de Gennes theory. Finally, we expand de Gennes melting theory to 3'5' pulling and compare the predictions with experimental results.

The structure of DNA overstretched from the 5'5' ends differs from the structure of DNA overstretched from the 3'3' ends.

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.