Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Mammalian orthologues of a yeast regulator of nonsense transcript stability.

All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1.

The 4-hydroxy metabolite of 17 beta-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbon-receptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 1B1. To determine the relationship of the human P450 1B1 gene product and E2 4-hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4- and 2-hydroxylation of E2 with Km values of 0.71 and 0.78 microM and turnover numbers of 1.39 and 0.27 nmol product min-1.nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 1B1 and P450 1A1 mRNA levels, and caused increased rates of 2-, 4-, 6 alpha-, and 15 alpha-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2- and 4-hydroxylation were approximately equal, emphasizing the significance of the low Km P450 1B1-component of E2 metabolism. These studies demonstrate that human P450 1B1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.

The pfmdr1 gene of Plasmodium falciparum confers cellular resistance to antimalarial drugs in yeast cells.

The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial. pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family. We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs. Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed. Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells. Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells. The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P. falciparum abolished the capacity of this protein to confer drug resistance. Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations.

Mammalian Sug1 and c-Fos in the nuclear 26S proteasome.

In a search for regulatory proteins that interact with the leucine zipper motif of c-Fos in the yeast two-hybrid screen, we have identified a protein (FZA-B) that has extensive sequence similarity to SUG1 of Saccharomyces cerevisiae. Here we show that FZA-B can functionally substitute for SUG1 in yeast and that FZA-B interacts with Fos proteins in vitro through their leucine zippers. In rat liver and in HeLa cells, FZA-B is present in the 26S proteasome complex, as is c-Fos. Immobilized antibody raised against an FZA-B-specific peptide depleted peptidase activity, proteasomal proteins, FZA-B, and c-Fos from a 26S proteasome preparation. FZA-B is found predominantly in the nuclear fraction of COS cells expressing an FZA-B transgene and in the nuclear 26S proteasome of HeLa cells. We conclude that FZA-B is the mammalian homolog of SUG1 (mSug1) and that it is present in the nuclear 26S proteasome of cells. Our results suggest that mSug1 may be involved in the degradation of c-Fos and other transcription factors.

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

In vitro reconstitution of human replication factor C from its five subunits.

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.

Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis.

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.

Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.

Movement of yeast cortical actin cytoskeleton visualized in vivo.

Fusion proteins between the green fluorescent protein (GFP) and the cytoskeleton proteins Act1p (actin), Sac6p (yeast fimbrin homolog), and Abp1p in budding yeast (Saccharomyces cerevisiae) localize to the cortical actin patches. The actin fusions could not function as the sole actin source in yeast, but fusions between the actin-binding proteins Abp1p and Sac6p complement fully the phenotypes associated with their gene deletions. Direct observation in vivo reveals that the actin cortical patches move. Movement of actin patches is constrained to the asymmetric distribution of the patches in growing cells, and this movement is greatly reduced when metabolic inhibitors such as sodium azide are added. Fusion protein-labeled patches are normally distributed during the yeast cell cycle and during mating. In vivo observation made possible the visualization of actin patches during sporulation as well.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.