Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus

The 2.15-A structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday junction complex is proposed, based on the available functional and structural data.     

A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.     

Stimulation of homologous recombination in plants by expression of the bacterial resolvase RuvC

Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.     

RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage. Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands. This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands. In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals. These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction.     

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.     

T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs.

We have formed four-arm branched DNA junctions that contain no more than a single base pair of branch migratory freedom. Recently, we have shown that these Holliday junction analogs have twofold symmetric protection patterns in solution when probed with hydroxyl radicals: two opposite strands of one junction show extensive protection near the branch point, while the other pair of opposite strands is virtually as susceptible as a double helix. In a different junction, the hydroxyl radical protection pattern is reversed. These patterns suggest that a crossover-isomer bias exists in these molecules and that the protected strands form the crossover between helices. Here, we examine the cleavage pattern of these structures when they are resolved by T4 endonuclease VII. Junctions are formed from a single shamrock-shaped molecule, which contains 5', 3', or internal labels. The enzyme shows a preference for resolving these modified junctions at sites near those protected from hydroxyl radicals. This result suggests that only crossover strands in a Holliday junction are cleaved, and thus an odd number of crossover isomerizations must occur when flanking markers are exchanged.     

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.     

Identification of core amino acids stabilizing rhodopsin

Rhodopsin is the only G protein-coupled receptor (GPCR) whose 3D structure is known; therefore, it serves as a prototype for studies of the GPCR family of proteins. Rhodopsin dysfunction has been linked to misfolding, caused by chemical modifications that affect the naturally occurring disulfide bond between C110 and C187. Here, we identify the structural elements that stabilize rhodopsin by computational analysis of the rhodopsin structure and comparison with data from previous in vitro mutational studies. We simulate the thermal unfolding of rhodopsin by breaking the native-state hydrogen bonds sequentially in the order of their relative strength, using the recently developed Floppy Inclusion and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader, A. J., Kuhn, L. A. & Thorpe, M. F. (2001) Proteins 44, 150-165]. Residues most stable under thermal denaturation are part of a core, which is assumed to be important for the formation and stability of folded rhodopsin. This core includes the C110-C187 disulfide bond at the center of residues forming the interface between the transmembrane and the extracellular domains near the retinal binding pocket. Fast mode analysis of rhodopsin using the Gaussian network model also identifies the disulfide bond and the retinal ligand binding pocket to be the most rigid region in rhodopsin. Experiments confirm that 90% of the amino acids predicted by the FIRST method to be part of the core cause misfolding upon mutation. The observed high degree of conservation (78.9%) of this disulfide bond across all GPCR classes suggests that it is critical for the stability and function of GPCRs.     

Evidence that eosinophils catalyze the bromide-dependent decarboxylation of amino acids

Human eosinophils from subjects with or without myeloperoxidase (MPO) deficiency and guinea pig eosinophils are able to decarboxylate L- alanine in the presence of the cationic detergent cetyltrimethylammonium bromide (CTAB) but not in the presence of the nonionic detergent Triton X-100. Instead, both normal human neutrophils and guinea pig neutrophils decarboxylate L-alanine in the presence of either detergent. When the non-bromide-containing cationic detergent cetyltrimethylammonium hydroxide (CTAOH) is used instead of CTAB, the eosinophils from MPO-deficient subjects are unable to decarboxylate L- alanine. Decarboxylation occurs with the combination CTAOH-Br-, but not with the combinations CTAOH-I-, CTAOH-CI-, or CTAOH-F-. Bromide in the absence of CTAOH does not promote decarboxylation. Triton X-100 and deoxycholate are much less effective in promoting decarboxylation in the presence of bromide. L-Lysine and L-aspartic acid are decarboxylated to a considerably lower rate than L-alanine in the presence of CTAOH and Br-. It is concluded that the eosinophils can catalyze the bromide-dependent decarboxylation of the apolar amino acid L-alanine in the presence of a cationic detergent.     

Identification of amino acids in Marburg virus VP40 that are important for virus-like particle budding

The matrix protein VP40 of Marburg virus promotes the formation and release of virus-like particles (VLPs). Marburg virus VP40 interacts with cellular Tsg101 via its L domain motif; however, mutation of this motif does not affect VLP budding or the accumulation of VP40 in multivesicular bodies (MVBs), which are platforms for virus particle formation. To identify regions of Marburg virus VP40 that are important for VLP budding, we examined deletion mutants and alanine-scanning mutants at the N- and C-terminus of VP40 for their involvement in VLP budding. VLPs were not detected in the presence of alanine-replacement mutants at Ile39 and Thr40, and the level of VLP budding for the alanine mutant at Asn297 was decreased. Moreover, these mutants did not accumulate in MVBs. Our results suggest the involvement of a novel host factor(s) in VLP budding and VP40 transport to MVBs.     

Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1.

ZO-1 is a 210- to 220-kDa peripheral membrane protein associated with the cytoplasmic surface of the epithelial tight junction. Because ZO-1 may interact with other unidentified tight junction proteins, we have looked for other polypeptides that bind to ZO-1. A 160-kDa polypeptide was identified that coimmunoprecipitates with ZO-1 from detergent extracts of metabolically labeled Madin-Darby canine kidney (MDCK) cells. This polypeptide appears to be distinct from ZO-1, rather than a degradation product, by several criteria. It lacks ZO-1 epitopes recognized by both monoclonal antibodies and a polyclonal serum to ZO-1, since it is not detectable in immunoblots of either whole cell extracts or ZO-1 immunoprecipitates. Also, it exhibits a peptide map different from that of ZO-1 on one-dimensional "Cleveland gels." Moreover, because the kinetics of appearance of newly synthesized 160-kDa polypeptide in anti-ZO-1 immunoprecipitates is much slower than that of ZO-1, its presence in immunoprecipitates cannot be simply explained by degradation of ZO-1 during cell lysis. Like ZO-1, the 160-kDa polypeptide seems to be a cytoplasmic peripheral membrane protein. It cannot be labeled by two different cell surface labeling reagents. It can be extracted from the membrane by high salt concentration in the absence of detergents. As expected for a protein complex, the 160-kDa polypeptide and ZO-1 turn over with similar kinetics. We propose that the 160-kDa polypeptide is a component of the tight junction.     

Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.

An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.     

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.     

CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein

Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ((5)S and (53)S) and at least three minor phosphorylation sites ((4)S, (7)S, and (59)T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of (5)S and (53)S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19-30 and containing (26)S and (27)S), it phosphorylated (26)S efficiently when this peptide contained a chemically synthesized phospho-(27)S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclin-dependent and Cdc7 kinases is essential for the initiation of DNA replication.     

A study of plasma free amino acid levels. V. Correlations among the amino acids and between amino acids and some other blood constituents

Possible relationships among the amino acids were examined by determining the correlation between their fasting plasma levels in 136 children and 193 adults. Leucine and isoleucine values were the most highly correlated. Threonine, serine, and glycine were significantly related in boys, girls, and men, but there was no correlation between threonine and glycine levels in women. A group of 11 amino acids appeared to be highly interrelated: These were alanine, asparagine, glutamine, valine, isoleucine, leucine, methionine, tyrosine, phenylalanine, proline, and lysine. Possible correlations between the individual amino acid levels and hemoglobin, blood glucose, plasma cholesterol, and uric acid levels were also examined. There were no highly significant relationships between amino acid levels and the levels of any of them.     

Identification of the iron-sulfur center in trimethylamine dehydrogenase.

Trimethylamine dehydrogenase [trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7] from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*). The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure. Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type. Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes [Fe4S*4(S2-o-xylyl)2]n2n- and [Fe4S*4(SC6H4OMe)4]2-, which were identified by absorption spectra. These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster [Fe4S*4(S-Cys)4].     

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.