Person:

Prentiss, Mara

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(\mu)M, 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.

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.

We demonstrate that buffer-gas cooling combined with laser ablation can be used to create coherent optical media with high optical depth and low Doppler broadening that offers metastable states with low collisional and motional decoherence. Demonstration of this generic technique opens pathways to coherent optics with a large variety of atoms and molecules. We use helium buffer gas to cool (^{87}Rb) atoms to below (7 K) and slow atom diffusion to the walls. Electromagnetically induced transparency in this medium allows for (50%) transmission in a medium with initial optical depth (D>70) and for slow pulse propagation with large delay-bandwidth products. In the high-(D) regime, we observe high-contrast spectrum oscillations due to efficient four-wave mixing.

RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.

This paper describes an empirical model of polymer dynamics, based on the agitation of millimeter-sized polymeric beads. Although the interactions between the particles in the macroscopic model, and those between the monomers of molecular-scale polymers, are fundamentally different, both systems follow the Worm-Like Chain theory.

We demonstrate a macroscopic magnetic guide for cold atom interferometry, where the magnetic guiding field is generated by a symmetrical array of racetrack coils of copper tape. This system represents a conceptual advance over previous guided atom interferometers based on nonsymmetrical geometries because the symmetry provides a much lower magnetic field curvature per fixed length than equivalent nonsymmetrical geometries, permitting a decrease in system length without increasing the decoherence rate associated with field curvature. We realized a magnetic guide a few cm away from each coil, where smooth translation of the guided atoms is achieved by changing the currents in second array of the multiple-conductor tape.

RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.

Accurate sequence dependent pairing of single-stranded DNA (ssDNA) molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

Long non-coding RNAs (lncRNAs) are prominently associated with chromosomes in an ever-increasing diversity of roles. To provide further insight into the potential nature of these associations, we have explored, for the first time, the interaction of long single-stranded (ss) RNAs with cognate homologous double-stranded (ds) DNA in vitro. Using magnetic tweezers, we measured the effects of ssRNA on force extension curves for dsDNA. We observe that the presence of ssRNA impedes the extension of dsDNA, specifically at low forces, dependent on homology between the RNA and DNA species, and dependent on ssRNA lengths (≥1 kb). The observed effect also depends on the concentration of ssRNA and is abolished by overstretching of the dsDNA. These findings show that significant homologous contacts can occur between long ssRNA and dsDNA in the absence of protein and that these contacts alter the mechanical properties of the dsDNA. We propose that long ssRNA interacts paranemically with long dsDNA via periodic short homologous interactions, e.g. mediated by RNA/DNA triplex-formation, and that dsDNA extension is impeded by formation of RNA secondary structure in the intervening unbound regions. Analogous interactions in vivo would permit lncRNAs to mediate the juxtaposition of two or more DNA regions on the same or different chromosomes.

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.