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.     

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.     

Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein.

The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions. Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution. The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end. There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP. During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected.     

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 four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.     

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.     

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.     

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.     

Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.     

Genetic recombination in Escherichia coli: Holliday junctions made by RecA protein are resolved by fractionated cell-free extracts.

Escherichia coli RecA protein catalyzes reciprocal strand-exchange reactions between duplex DNA molecules, provided that one contains a single-stranded gap or tail, to form recombination intermediates containing Holliday junctions. Recombination reactions are thought to occur within helical RecA-nucleoprotein filaments in which DNA molecules are interwound. Structures generated in vitro by RecA protein have been used to detect an activity from fractionated E. coli extracts that resolves the intermediates into heteroduplex recombinant products. Resolution occurs by specific endonucleolytic cleavage at the Holliday junction. The products of cleavage are characteristic of patch and splice recombinants.     

Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.     

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1–S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2′, R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell–cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2′ cleavage site within S2 793-KPTKR-797 (S2′), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2′ position allowed trypsin-independent cell–cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1–S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2′), acting in concert with the S1–S2 cleavage site to mediate membrane fusion and virus infectivity.     

The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2. 1 A. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson-Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4 degrees as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3'-C x G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.     

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.     

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.     

On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli.

The renaturation of complementary DNA strands by the recA protein of Escherichia coli has been found to exhibit the following features. (i) Optimal renaturation occurs at recA protein levels below that required to saturate the DNA strands; saturating amounts of recA protein significantly reduce the rate of reaction. (ii) The reaction proceeds in the absence of a nucleotide cofactor but is markedly stimulated by ATP in the presence of 10 mM Mg2+. A similar stimulation occurs in the absence of ATP when the Mg2+ concentration is increased from 10 mM to 30-40 mM. (iii) Both the ATP-stimulated and the Mg2+-stimulated reactions follow apparent first-order kinetics. These results, taken together with the known effects of ATP and Mg2+ on the state of aggregation of recA protein, suggest that the association of recA monomers may play an important role in recA protein-promoted DNA renaturation.     

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific Pbeta domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser(77)-Ala(78) that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology.