Expression of alpha3, beta3 and gamma1 GABA(A) receptor subunit messenger RNAs in visual cortex and lateral geniculate nucleus of normal and monocularly deprived monkeys

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is "invisible", showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (x10(-5)), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.     

Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.     

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.     

Promoter specificity determinants of T7 RNA polymerase

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.     

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.     

Formation of a single base mismatch impedes spontaneous DNA branch migration

DNA branch migration, a process whereby two homologous DNA duplexes exchange strands, is an essential component of genetic recombination. Models for homologous recombination have invoked spontaneous branch migration as one mechanism for the generation of large regions of heteroduplex DNA. During recombination, two homologous parental duplexes that contain similar, but not identical, sequences are paired and undergo strand exchange. An important issue is whether spontaneous branch migration is capable of traversing sequence heterology such as mismatches, insertions and deletions. We use a model four-strand system to examine the effect of mispaired or unpaired bases on branch migration. The assay consists of annealing two short duplexes having defined sequence heterologies. Following annealing, a Holliday junction is formed that is free to branch migrate. Our results demonstrate that a single base mismatch, insertion or deletion is sufficient to pose a substantial barrier to spontaneous branch migration. In the presence of magnesium, branch migration through such sequence heterologies is almost completely blocked. Others have shown that non-mobile four-way junctions undergo a dramatic shift in conformation in the presence of magnesium. Our data suggest that a similar transition occurs for the mobile Holliday junction. We also discuss how proteins may facilitate branch migration through sequence heterologies in vivo.     

Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe

The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products. Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria. We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1. The YDC2 protein has been cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme. YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer. The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange. The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S. cerevisiae and S. pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.     

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.     

Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion

E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors. In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues. The nicked duplex products are repaired by the action of DNA ligase. Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack. The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.     

Twin-twin transfusion syndrome: the challenge of etiology-based management decisions

Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday junction. The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules. It also reveals directly for the first time the structure of the Holliday junction. A single RuvA tetramer is bound to one face of a junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation. Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner. The open structure of the junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.     

Analysis of substrate specificity of the RuvC holliday junction resolvase with synthetic Holliday junctions

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.     

Formation of RuvABC-Holliday junction complexes in vitro

In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair. RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process. Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC). Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected. In the absence of RuvB, RuvAC-junction complexes were observed. Complex formation was not facilitated by duplex DNA. The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction.     

Interaction of the resolving enzyme YDC2 with the four-way DNA junction

Holliday junctions (four-way DNA junctions), formed during homologous recombination, are bound and resolved by junction-specific endonucleases to yield recombinant duplex DNA products. The junction-resolving enzymes are a structurally diverse class of proteins that nevertheless have many properties in common; in particular a high structure specificity for binding and metal-dependent, (frequently) sequence-specific cleavage activity. In Saccharomyces cerevisiae, the enzyme CCE1 is necessary for the resolution of recombining mitochondrial genomes, and in Schizosaccharomyces pombe the homologous protein YDC2 is thought to have a similar function. We have generated an inactive mutant of YDC2, D226N, that retains structure-specific junction binding and have analysed the interaction of this protein with the four-way DNA junction. YDC2 binds the four-way junction in two specific complexes (I and II), unfolding the stacked X-structure into a conformation where the arms extend to the four corners of a square. This structure is reminiscent of that of the free junction in the absence of metal ions and of the structures imposed on the Holliday junction by CCE1 and RuvA. DNase I probing reveals footprints specific for complexes I and II which extend from the junction centre on all four arms. No protection is observed with the small, hydrophobic probe DMS.     

Functional analysis of coordinated cleavage in V(D)J recombination

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.     

Preference of the recombination sites involved in the formation of extrachromosomal copies of the human alphoid Sau3A repeat family

The human alphoid Sau3A repetitive family DNA is one of the DNA species that are actively amplified to form extrachromosomal circular DNA in several cell lines. The circularization takes place between two of the five approximately 170 bp subunits with an average of 73.1% homology as well as between identical subunits. To investigate the nature of the recombination reaction, we cloned and analyzed the subunits containing recombination junctions. Analysis of a total of 68 junctions revealed that recombination had occurred preferentially at four positions 10-25 (A), 40-50 (B), 85-90 (C) and 135-160 (D) in the 170bp subunit structure. Two regions (B and C) were overlapped with the regions with higher homology between subunits, while other two regions (A and D) cannot be explained solely by the regional homology between the subunits. These regions were located at both junctions of the nucleosomal and the linker region, and overlapped with the binding motifs for alpha protein and CENP-B. Approximately 90% of the recombination occurred between the subunits located next but one (+/- 2 shift), although the frequency of recombination between the adjoining subunits (+/- 1 shift) was approximately 10%.     

High resolution computed tomography (HRCT) of pulmonary infections in AIDS patients

The Escherichia coli RuvA and RuvB proteins mediate ATP-dependent branch migration of Holliday junctions during homologous genetic recombination. RuvA is a DNA-binding protein with high affinity for Holliday junctions, to which it directs RuvB (a DNA-dependent ATPase). Electron microscopic studies have shown that RuvB forms double hexameric rings on duplex DNA. To determine whether the rings are biologically active, the conditions required for their formation and activity have been analysed. The quaternary structure of RuvB appears to be dependent upon the binding of ATP, magnesium ions, and the presence of RuvA. In the presence of Mg2+ and ATP, RuvB forms hexamers; however, in the presence of Mg2+ alone, dodecamers were observed. Both forms of the protein are stable and have been isolated by gel filtration. Performed dodecamers and, to a lesser extent, hexamers assembled in the absence of DNA lack ATPase activity. Maximal ATPase activity was observed when RuvB assembled directly on DNA in the presence of Mg2+ and ATP. Moreover, under these conditions, a direct interaction between RuvB hexamers and tetramers of RuvA was observed.     

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli

Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA. A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange. In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure. In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene. Here, we describe the DNA binding properties of RusA. We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA. However, it does bind weakly to linear duplex DNA. Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions. The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.