Homologous recombination in real time: DNA strand exchange by RecA.

Homologous recombination, the exchange of strands between different DNA molecules, is essential for proper maintenance and accurate duplication of the genome. Using magnetic tweezers, we monitor RecA-driven homologous recombination of individual DNA molecules in real time. We resolve several key aspects of DNA structure during and after strand exchange. Changes in DNA length and twist yield helical parameters for the protein-bound three-stranded structure in conditions in which ATP was not hydrolyzed. When strand exchange was completed under ATP hydrolysis conditions that allow protein dissociation, a "D wrap" structure formed. During homologous recombination, strand invasion at one end and RecA dissociation at the other end occurred at the same rate, and our single-molecule analysis indicated that a region of only about 80 bp is actively involved in the synapsis at any time during the entire reaction involving a long ( approximately 1 kb) region of homology.

Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy.

Nucleosome positioning dictates eukaryotic DNA compaction and access. To predict nucleosome positions in a statistical mechanics model, we exploited the knowledge that nucleosomes favor DNA sequences with specific periodically occurring dinucleotides. Our model is the first to capture both dyad position within a few base pairs, and free binding energy within 2 k(B)T, for all the known nucleosome positioning sequences. By applying Percus's equation to the derived energy landscape, we isolate sequence effects on genome-wide nucleosome occupancy from other factors that may influence nucleosome positioning. For both in vitro and in vivo systems, three parameters suffice to predict nucleosome occupancy with correlation coefficients of respectively 0.74 and 0.66. As predicted, we find the largest deviations in vivo around transcription start sites. This relatively simple algorithm can be used to guide future studies on the influence of DNA sequence on chromatin organization.

Characterization of nucleosome unwrapping within chromatin fibers using magnetic tweezers.

Nucleosomal arrays fold into chromatin fibers and the higher-order folding of chromatin plays a strong regulatory role in all processes involving DNA access, such as transcription and replication. A fundamental understanding of such regulation requires insight into the folding properties of the chromatin fiber in molecular detail. Despite this, the structure and the mechanics of chromatin fibers remain highly disputed. Single-molecule force spectroscopy experiments have the potential to provide such insight, but interpretation of the data has been hampered by the large variations in experimental force-extension traces. Here we explore the possibility that chromatin fibers are composed of both single-turn and fully wrapped histone octamers. By characterizing the force-dependent behavior of in vitro reconstituted chromatin fibers and reanalyzing existing data, we show the unwrapping of the outer turn of nucleosomal DNA at 3 pN. We present a model composed of two freely-jointed chains, which reveals that nucleosomes within the chromatin fiber show identical force-extension behavior to mononucleosomes, indicating that nucleosome-nucleosome interactions are orders-of-magnitude smaller than previously reported and therefore can be overcome by thermal fluctuations. We demonstrate that lowering the salt concentration externally increases the wrapping energy significantly, indicative of the electrostatic interaction between the wrapped DNA and the histone octamer surface. We propose that the weak interaction between nucleosomes could allow easy access to nucleosomal DNA, while DNA unwrapping from the histone core could provide a stable yet dynamic structure during DNA maintenance.

Coexistence of twisted, plectonemic, and melted DNA in small topological domains.

DNA responds to small changes in force and torque by over- or undertwisting, forming plectonemes, and/or melting bubbles. Although transitions between either twisted and plectonemic conformations or twisted and melted conformations have been described as first-order phase transitions, we report here a broadening of these transitions when the size of a topological domain spans several kilobasepairs. Magnetic tweezers measurements indicate the coexistence of three conformations at subpicoNewton force and linking number densities ∼-0.06. We present a statistical physics model for DNA domains of several kilobasepairs by calculating the full partition function that describes this three-state coexistence. Real-time analysis of short DNA tethers at constant force and torque shows discrete levels of extension, representing discontinuous changes in the size of the melting bubble, which should reflect the underlying DNA sequence. Our results provide a comprehensive picture of the structure of underwound DNA at low force and torque and could have important consequences for various biological processes, in particular those that depend on local DNA melting, such as the initiation of replication and transcription.

Dynamics of RecA filaments on single-stranded DNA.

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA-ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA-ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.

Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules.

The human DNA repair protein RAD51 is the crucial component of helical nucleoprotein filaments that drive homologous recombination. The molecular mechanistic details of how this structure facilitates the requisite DNA strand rearrangements are not known but must involve dynamic interactions between RAD51 and DNA. Here, we report the real-time kinetics of human RAD51 filament assembly and disassembly on individual molecules of both single- and double-stranded DNA, as measured using magnetic tweezers. The relative rates of nucleation and filament extension are such that the observed filament formation consists of multiple nucleation events that are in competition with each other. For varying concentration of RAD51, a Hill coefficient of 4.3 +/- 0.5 is obtained for both nucleation and filament extension, indicating binding to dsDNA with a binding unit consisting of multiple (> or =4) RAD51 monomers. We report Monte Carlo simulations that fit the (dis)assembly data very well. The results show that, surprisingly, human RAD51 does not form long continuous filaments on DNA. Instead each nucleoprotein filament consists of a string of many small filament patches that are only a few tens of monomers long. The high flexibility and dynamic nature of this arrangement is likely to facilitate strand exchange.

Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule.

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.

Monte carlo simulations of protein assembly, disassembly, and linear motion on DNA.

We use Monte Carlo simulations to analyze the simultaneous interactions of multiple proteins to a long DNA molecule. We study the time dependence of protein organization on DNA for different regimes that comprise (non)cooperative sequence-independent protein assembly, dissociation, and linear motion. A range of different behaviors is observed for the dynamics, final coverage, and cluster size distributions. We observe that the DNA substrate is almost never completely covered by protein when taking into account only (non)cooperative binding, because gaps remain on the substrate that are smaller than the binding site size of the protein. Due to these gaps, the apparent binding size of a protein during noncooperative binding can be overestimated by up to 30%. During dissociation of cooperatively bound proteins, the dissociation curve can be exponentially shaped even when allowing only end-dependent dissociation. We discuss the potential of our method for the analysis of a number of single-molecule experiments, for example, the binding of the DNA-repair proteins RecA and Rad51 to DNA.

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.

Torque-limited RecA polymerization on dsDNA.

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.

Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion.

Recognition of 'foreign' DNA by Type I restriction-modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR2:HsdM2:HsdS1 (R2-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR1:HsdM2:HsdS1 (R1-complex). ATPS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R1-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R2-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of approximately 11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.

High flexibility of DNA on short length scales probed by atomic force microscopy.

The mechanics of DNA bending on intermediate length scales (5-100 nm) plays a key role in many cellular processes, and is also important in the fabrication of artificial DNA structures, but previous experimental studies of DNA mechanics have focused on longer length scales than these. We use high-resolution atomic force microscopy on individual DNA molecules to obtain a direct measurement of the bending energy function appropriate for scales down to 5 nm. Our measurements imply that the elastic energy of highly bent DNA conformations is lower than predicted by classical elasticity models such as the worm-like chain (WLC) model. For example, we found that on short length scales, spontaneous large-angle bends are many times more prevalent than predicted by the WLC model. We test our data and model with an interlocking set of consistency checks. Our analysis also shows how our model is compatible with previous experiments, which have sometimes been viewed as confirming the WLC.

The coiled-coil of the human Rad50 DNA repair protein contains specific segments of increased flexibility.

Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules. However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function. In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein. Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only. Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the local flexibility from high-resolution atomic force microscopy images. Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed. We discuss how this dynamic structural feature is integral to Rad50 function.