Kristi Hatch

Single Molecule Detection of Direct, Homologous, DNA/DNA Pairing

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.

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 λ 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 μm 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 β-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.

Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein

The rezipping force of two complementary DNA strands under tension has been measured in the presence of Escherichia coli single-stranded-binding proteins under salt conditions ranging from 10– to 400 mM NaCl. The effectiveness of the binding protein in preventing rezipping is strongly dependent on salt concentration and compared with the salt dependence in the absence of the protein. At concentrations less than 50 mM NaCl, the protein prevents complete rezipping of λ-phage on the 2-s timescale of the experiment, when the ssDNA is under tensions as low as 3.5 ± 1 pN. For salt concentrations greater than 200 mM NaCl, the protein inhibits rezipping but cannot block rezipping when the tension is reduced below 6 ± 1.8 pN. This change in effectiveness as a function of salt concentration may correspond to salt-dependent changes in binding modes that were previously observed in bulk assays.

Probing the mechanical stability of DNA in the presence of monovalent cations.

We examine the interaction between monovalent cations and DNA using several different assays that measure the stability of double-stranded DNA (dsDNA). The thermal melting of dsDNA and the mechanical separation of dsDNA into two single strands both depend on the stability of dsDNA with respect to ssDNA and are sensitive to the interstrand phosphate repulsion. We find that the experimentally measured melting temperatures and unzipping forces are approximately the same for all of the ions considered in this study. Likewise, the force required to transform B-DNA into the overstretched form is also similar for all of the ions. In contrast, for a given ion concentration, the force at which the overstretched state fully relaxes back to the canonical B-DNA form depends on the cation; however, for all cations, the overstretching force decreases with decreasing ion concentration, suggesting that this force is sensitive to screening. We observe a general trend for smaller ions to produce more efficient relaxation. Finally, for a given cation, the relaxation can also depend on the anion.