RuvA is a sliding collar that protects Holliday junctions from unwinding while promoting branch migration

The RuvAB proteins catalyze branch migration of Holliday junctions during DNA recombination in Escherichia coli. RuvA binds tightly to the Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with its cellular partner RuvB. The replication fork helicase DnaB catalyzes branch migration like RuvB but, unlike RuvB, is not dependent on RuvA for activity. In this study, we specifically analyze the function of RuvA by studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein-protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA.     

An effective mesoscopic model of double-stranded DNA

Watson and Crick’s epochal presentation of the double helix structure in 1953 has paved the way to intense exploration of DNA’s vital functions in cells. Also, recent advances of single molecule techniques have made it possible to probe structures and mechanics of constrained DNA at length scales ranging from nanometers to microns. There have been a number of atomistic scale quantum chemical calculations or molecular level simulations, but they are too computationally demanding or analytically unfeasible to describe the DNA conformation and mechanics at mesoscopic levels. At micron scales, on the other hand, the wormlike chain model has been very instrumental in describing analytically the DNA mechanics but lacks certain molecular details that are essential in describing the hybridization, nano-scale confinement, and local denaturation. To fill this fundamental gap, we present a workable and predictive mesoscopic model of double-stranded DNA where the nucleotides beads constitute the basic degrees of freedom. With the inter-strand stacking given by an interaction between diagonally opposed monomers, the model explains with analytical simplicity the helix formation and produces a generalized wormlike chain model with the concomitant large bending modulus given in terms of the helical structure and stiffness. It also explains how the helical conformation undergoes overstretch transition to the ladder-like conformation at a force plateau, in agreement with the experiment.     

Geometric features of the DNA double helix in the supercoiled state

The geometric features of the DNA molecule in the supercoiled state were considered. A model of the supercoiled structure of the DNA molecule was constructed; the model takes into account its natural helicity. The force factors arising in the molecule at various superhelix angles were calculated.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

Model for RuvAB-mediated branch migration of Holliday junctions

During RuvAB-mediated Holliday-junction migration two opposite arms of double-stranded DNA (dsDNA) are driven to translocate unidirectional by two respective ring-like hexameric RuvB proteins. However, how the RuvB protein, powered by ATP hydrolysis, drives unidirectional translocation of dsDNA is not clear. Here a model is presented for this mechanochemical-coupling mechanism. In the model, the unidirectional translocation is resulted from both the ATP hydrolysis-induced rotation (power stroke) of the RuvB subunits and the passage of the strong DNA binding from the previous to next RuvB subunits during the sequential ATPase activities around the ring. Using the model, the relationship between the power-stroke size, the step size of DNA translocation and the ratio of the rotational rate of DNA over that of RuvB relative to RuvA is predicted.     

Apical control of branch growth and angle in woody plants

Apical control is the inhibition of a lateral branch growth by shoots above it (distal shoots). If the distal shoots are cut off to remove apical control, the lateral branch can grow larger and may bend upwards. Apical control starts when new lateral buds grow after passing through a period of dormancy. Buds initially break and produce leaves, then apical control is exerted and the lower (proximal) laterals stop growing. Apical control also inhibits growth of large, old branches. Gravimorphism and restricted water and nutrient transport can inhibit branch growth, but they are not primary mechanisms of apical control. Apical control may reduce branch photosynthesis. Under apical control allocation of branch-produced assimilate to the stem is relatively high, so low assimilates in the branch may limit branch growth even though hormone levels are adequate for growth. Hormones appear to be involved in apical control, but it is not known how. One role of hormones may be to maintain the strength of the stem sink for branch-produced assimilate. Upward bending of a woody branch after release from apical control requires both new wood production and production of wood cells that can generate an upward bending moment. Apical control inhibits radial growth of branches and, in some species, may regulate the production of wood with an upward bending moment.     

p53 blocks RuvAB promoted branch migration and modulates resolution of Holliday junctions by RuvC

The Holliday junction is the central intermediate in homologous recombination. Branch migration of this four-stranded DNA structure is a key step in genetic recombination that affects the extent of genetic information exchanged between two parental DNA molecules. Here, we have constructed synthetic Holliday junctions to test the effects of p53 on both spontaneous and RuvAB promoted branch migration as well as the effect on resolution of the junction by RuvC. We demonstrate that p53 blocks branch migration, and that cleavage of the Holliday junction by RuvC is modulated by p53. These findings suggest that p53 can block branch migration promoted by proteins such as RuvAB and modulate the cleavage by Holliday junction resolution proteins such as RuvC. These results suggest that p53 could have similar effects on eukaryotic homologues of RuvABC and thus have a direct role in recombinational DNA repair.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Structural polymorphism of liquid-crystalline dispersions of double-stranded DNA complexed with synthetic polycations

Linear double-stranded DNA molecules interact with positively charged polyconidine molecules in aqueous salt solutions to yield liquid-crystalline dispersions (LCDs) with a mean particle diameter of ～6000 Å. The packing density of (DNA-polycation) complexes differs among LCD particles formed at different ionic strengths. X-ray data on the liquid-crystalline phases of (DNA-polyconidine) complexes formed under different conditions were compared with a phase diagram, reflecting polymorphism of liquid crystals of linear double-stranded DNA. It was shown that LCD was hexagonal at 0.15 M ≤ C _NaCl < 0.4 M and cholesteric at 0.4 M ≤ C _NaCl < 0.55 M. Cholesteric LCD displayed abnormal optical activity in the circular dichroism spectrum. A similar situation was observed with poly(2,5-ionene), another polycation differing in chemical structure from polyconidine. The results demonstrated structural polymorphism of (DNA-polycation) LCDs. It was assumed that the packing mode of (DNA-polycation) complexes in LCD particles can be regulated by changing NaCl concentration. The mechanism generating the cholesteric liquid-crystalline state of DNA in a narrow range of NaCl concentrations is discussed.     

Rapid and reliable dideoxy sequencing of double-stranded DNA

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M 13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.     

Liquid-crystalline dispersions formed by the complexes of linear double-stranded DNA molecules with dendrimers

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.     

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination

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Functional significance of octameric RuvA for a branch migration complex from Thermus thermophilus

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.     

From strand exchange to branch migration; bypassing of non-homologous sequences by human Rad51 and Rad54

Rad51 and Rad54 play crucial roles during homologous recombination. The biochemical activities of human Rad51 (hRad51) and human Rad54 (hRad54) and their interactions with each other are well documented. However, it is not known how these two proteins work together to bypass heterologous sequences; i.e. mismatched base pairs, during homologous recombination. In this study, we used a fluorescence resonance energy transfer assay to monitor homologous recombination processes in real time so that the interactions between hRad54 and hRad51 during DNA strand exchange and branch migration, which are two core steps of homologous recombination, could be characterized. Our results indicate that hRad54 can facilitate hRad51-promoted strand exchange through various degrees of mismatching. We propose that the main roles of hRad51 in homologous recombination is to initiate the homology recognition and strand-exchange steps and those of hRad54 are to promote efficient branch migration, bypass potential mismatches and facilitate long-range strand exchanges through branch migration of Holliday junctions.     

DNA recognition by structure-selective nucleases

The nucleases discussed in this review show little sequence specificity but instead recognize certain structural features of their respective DNA substrates. The level of their structural selectivity ranges from simple discrimination between single- and double-stranded DNA (nucleases P1 and S1), the recognition of helical parameters like groove width and flexibility (DNase I), the recognition of helical distortions caused by abasic sites (exonuclease III, HAP1), to the recognition of specialized structures like flap DNA (5′-nucleases of eukaryotes, phages, and eubacterial DNA polymerases) and four-way junctions (T4 endonuclease VII, RuvC). The discussion is focused on the structural basis of the recognition process. In most cases the available x-ray structures of the nucleases and/or their DNA complexes have revealed the presence of structural motifs explaining the observed structural selectivity. © 1998 John Wiley & Sons, Inc. Biopoly 44: 405–421, 1997     

The inherent properties of DNA four-way junctions: comparing the crystal structures of holliday junctions

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl₂. Incubation of 0 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg²⁺ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0.15 M NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.     

Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data

An overview of published approaches for the metabolic flux control analysis of branch points revealed that often not all fundamental constraints on the flux control coefficients have been taken into account. This has led to contradictory statements in literature on the minimum number of large perturbation experiments required to estimate the complete set of flux control coefficients C(J) for a metabolic branch point. An improved calculation procedure, based on approximate Lin-log reaction kinetics, is proposed, providing explicit analytical solutions of steady state fluxes and metabolite concentrations as a function of large changes in enzyme levels. The obtained solutions allow direct calculation of elasticity ratios from experimental data and subsequently all C(J)-values from the unique relation between elasticity ratio's and flux control coefficients. This procedure ensures that the obtained C(J)-values satisfy all fundamental constraints. From these it follows that for a three enzyme branch point only one characterised or two uncharacterised large flux perturbations are sufficient to obtain all C(J)- values. The improved calculation procedure is illustrated with four experimental cases.