Single-molecule study of RuvAB-mediated Holliday-junction migration

Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximately 6-kb DNA molecules of identical sequence, and we measured the migration rate at 37 degrees C and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximately 43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).     

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.     

Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA-RuvB protein complex

The Escherichia coli RuvA-RuvB complex promotes branch migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA-RuvB promotes branch migration by driving helical rotation of the DNA. To clarify the RuvA-RuvB-mediated branch migration mechanism in more detail, we observed DNA rotation during Holliday junction branch migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA-RuvB-mediated branch migration at various ATP concentrations. The data provided a K(m) value of 65 microM and a V(max) value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA-RuvB-mediated branch migration, but also opens the possibility of elucidating the branch migration mechanism in detail.     

Holliday junction dynamics and branch migration: single-molecule analysis

The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.     

RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling

Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre-steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.     

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.     

Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-A resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.     

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.     

The kinetics of spontaneous DNA branch migration.

An important step in genetic recombination is DNA branch migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous branch migration as a function of temperature and ionic conditions. The branch migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of branch migration is exceedingly sensitive to the type of metal ions present. In magnesium, branch migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, branch migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the branch migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the branch point plays a key role in determining the rate of spontaneous DNA branch migration. We discuss the role of proteins in promoting the branch migration step during homologous recombination.     

Processive movement of single 22S dynein molecules occurs only at low ATP concentrations

We have analyzed the movement of single 22S dynein molecules from Tetrahymena cilia by using a nanometer measuring system equipped with optical tweezers. Statistical analysis proved that a single molecule of 22S dynein can move processively and develop force at low concentrations of ATP (<20 microM). The maximum force was approximately 4.7 pN, and the force-velocity curve was convex down. During force development, dynein molecules showed stepwise displacement of approximately 8 nm and frequently exhibited backward steps of approximately 8 nm. At higher concentrations of ATP (>/=20 microM) single molecules of 22S dynein were not observed to move processively. Twenty-two S dynein seems to switch over from a processive mode to a nonprocessive mode, sensing a subtle change of ATP concentrations. These observations indicate that the processivity, maximum force, and step size of dynein are similar to those of kinesin, but the ATP concentration-dependence, force-velocity relationship, and backward steps are clearly distinct from kinesin.     

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork ( approximately 400 bp/s), its unwinding rate extrapolated to zero force is much slower ( approximately 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.     

Angioplasty techniques for stenoses involving coronary artery bifurcations

Branch occlusion during coronary angioplasty is an infrequent but potentially serious complication. The overall incidence of branch occlusion during dilatation of a primary vessel is 5%. Branch vessels most jeopardized by dilatation generally have a complex plaque that not only involves the target vessel but also extends into the origin of the branch vessel. Branches free of pathology at their origin generally have an exceedingly low incidence of occlusion during adjacent balloon dilatation. Side branches at risk for occlusion should be "protected" if the branch vessel is of an important size that could be dilated with a conventional dilatation catheter. The advent of lower profile dilatation catheters and guidewires has provided an opportunity to introduce several pieces of dilatation hardware into the coronary system through a single guiding catheter. Several techniques are described for both "protecting" and dilating side branches, either simultaneously or secondarily, after balloon dilatation of a primary vessel.     

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.     

SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA.

The ruv operon is induced by treatments that damage DNA and is regulated by the LexA repressor. It encodes two proteins, RuvA and RuvB, that are involved in DNA repair, recombination in RecE and RecF pathways, and mutagenesis. RuvB protein was previously purified and has ATP-binding activity and weak ATPase activity. To study the biochemical properties of RuvA and its interaction with RuvB, we purified RuvA protein to near homogeneity from an over-producing strain. RuvA bound more efficiently to single-stranded DNA than to double-stranded DNA. RuvA bound to DNA greatly enhanced the ATPase activity of RuvB; the enhancing effect of various forms of DNA was in the order of supercoiled DNA greater than single-stranded DNA greater than linear double-stranded DNA. UV irradiation further enhanced the ATPase stimulatory effect of supercoiled DNA dose dependently. The RuvA-RuvB complex has an activity that renatures the cruciform structure in supercoiled DNA. From these experiments and previous work, we infer that the RuvA-RuvB complex may promote branch migration in recombination and may correct irregular structures in DNA, such as cruciforms and hairpins, to facilitate DNA repair using ATP as the energy source.     

Origin and Direction of Replication of Bacteriophage 186 DNA

Intracellular bacteriophage 186 DNA replicates as a single-branched circle during the first round of replication. The free end of the branch is located at a unique position with respect to phage 186 DNA base sequence, and this point should, therefore, correspond to the origin of DNA replication. The position of the growing point has been mapped at various degrees of replication, and found to move unidirectionally from left to right with respect to the denaturation map of phage 186 DNA.A small proportion of the replicating molecules have two linear branches connected to the circle at two different branch points. These structures are consistent with two separate initiations from the same origin, again, with a unidirectional mode of replication. Branch points frequently have a single-stranded connection between the circle and the branch; significant numbers of branch points also possess an extra short single-stranded "whisker" protruding out of the branch point.     

Slow cruciform transitions in palindromic DNA.

Extrusion of cruciform structures in self-complementary regions of DNA is known to be favored by negative supercoiling of DNA. We show here that, in moderately supercoiled DNA, cruciform extrusion is a very slow process. In plasmid pUC7 DNA, with a 48-base-pair palindrome, the half-time of extrusion at 50 degrees C is typically several hours; rates are even slower at lower temperature. The rates increase significantly with increasing DNA supercoiling but are only slightly faster in DNA species with much longer palindromes. The reabsorption of cruciform arms is also very slow. The equilibrium between cruciform and regular DNA structures is sensitive to changes in the linking number. Measurement of this equilibrium leads to an estimate of 18 kcal/mol (75.3 kJ/mol) for the free energy required to generate a cruciform structure. In bacterial cells, cruciform DNA may be rare, even when it is thermodynamically favored, because of its slow formation.     

Nucleosome positioning is determined by the (H3-H4)2 tetramer.

It is demonstrated that the histone (H3-H4)2 tetramer can find specific positions on DNA, even in the absence of other histones. Purified histone (H3-H4)2 tetramers were reconstituted onto 208-base-pair (bp) DNA molecules containing a nucleosome-positioning sequence by using salt-gradient dialysis. The stoichiometry of histone tetramer to DNA was shown to be 1:1. Digestion with micrococcal nuclease led to formation of protected DNA fragments of approximately 73 bp. Cleavage of the 73-bp DNA with restriction enzymes produced a small set of defined bands, demonstrating positioning of the (H3-H4)2 tetramer on DNA. Analysis of the restriction digests shows that the 73-bp DNA corresponds mainly to two fragments, one lying on either side of the pseudo-dyad axis of the major position adopted by complete histone octamers on this DNA. This result means that a single (H3-H4)2 histone tetramer can fold approximately 146 bp of DNA with the same positioning as the complete octamer but that a region near the pseudo-dyad is only weakly protected against micrococcal nuclease attack in the absence of histones H2A and H2B.     

Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli.

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.     

Dynamics of the leading process,nucleus, and Golgi apparatus of migrating cortical interneurons in living mouse embryos

Precisely arranged cytoarchitectures such as layers and nuclei depend on neuronal migration, of which many in vitro studies have revealed the mode and underlying mechanisms. However, how neuronal migration is achieved in vivo remains unknown. Here we established an imaging system that allows direct visualization of cortical interneuron migration in living mouse embryos. We found that during nucleokinesis, translocation of the Golgi apparatus either precedes or occurs in parallel to that of the nucleus, suggesting the existence of both a Golgi/centrosome-dependent and -independent mechanism of nucleokinesis. Changes in migratory direction occur when the nucleus enters one of the leading process branches, which is accompanied by the retraction of other branches. The nucleus occasionally swings between two branches before translocating into one of them, the occurrence of which is most often preceded by Golgi apparatus translocation into that branch. These in vivo observations provide important insight into the mechanisms of neuronal migration and demonstrate the usefulness of our system for studying dynamic events in living animals.