Note: A simple model for thermal management in solenoids.

We describe a model of the dynamical temperature evolution in a solenoid winding. A simple finite element analysis is calibrated by accurately measuring the thermally induced resistance change of the solenoid, thus obviating the need for accurate knowledge of the mean thermal conductivity of the windings. The model predicts quasi thermal runaway for relatively modest current increases from the normal operating conditions. We demonstrate the application of this model to determine the maximum current that can be safely applied to solenoids used for helium spin-echo measurements.

Measurement of the phason dispersion of misfit dislocations on the Au(111) surface.

We report measurements of the acoustic and optical phason dispersion curves associated with the lattice of partial dislocations on the reconstructed (111) surface of gold. Our measurements of these low energy (<0.5 meV) weakly dispersive modes have been enabled by the very high resolution of the novel helium spin-echo technique. The results presented here constitute the first measurement of the phason dispersion of misfit dislocations, and possibly of excitations associated with any type of crystalline dislocations.

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes.

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.

Expression and cytoplasmic localisation of deoxyuridine triphosphate pyrophosphatase encoded by a human endogenous retrovirus.

Many lentiviruses encode a dUTPase which may protect against toxic misincorporation of dUTP into cDNA during reverse transcription. However, the primate lentiviruses HIV and SIV do not express a dUTPase. Significantly, the host genomes of these lentiviruses contain a multicopy endogenous retrovirus which is absent in non-primate genomes. In humans, this endogenous retrovirus is known as HERV-K and encodes a potential dUTPase sequence. Previously, we have suggested that HIV infection is complemented by a cytosolic dUTPase derived from the dUTPase gene encoded by HERV-K. This study demonstrates expression of HERV-K dUTPase transcripts and protein in human cell lines using RT-PCR and western blot analysis. Immunocytochemistry showed that HERV-K dUTPase was predominantly located in cell cytoplasm when transiently expressed in COS-1 cells. These data provide substantiation and support for the hypothesis above and is the first documentation of expression of an enzyme of nucleotide metabolism expressed by an endogenous retrovirus.

Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target.

dUTP pyrophosphatase catalyses hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and inorganic pyrophosphate (PPi). Elimination of dUTP is vital since its misincorporation into DNA by DNA polymerases can initiate a damaging iterative repair and misincorporation cycle, resulting in DNA fragmentation and cell death. The anti-tumour activity of folate agonists and thymidylate synthase inhibitors is thought to rely on dUTP misincorporation. Furthermore, retroviral cDNA production may be particularly susceptible to the effects of dUTP misincorporation by virtue of the error-prone nature of reverse trans criptase. Consequently, dUTPase activity is an ideal point of intervention in both chemotherapy and anti-retroviral therapy. In particular, the dUTPase encoded by a human endogenous retrovirus (HERV-K) has been suggested to complement HIV infection and so is an attractive target for specific inhibition. Hence, we used site photoaffinity labelling, site-directed mutagenesis and molecular modelling to assign catalytic roles to the conserved amino acid residues in the active site of the HERV-K dUTPase and to identify structural differences with other dUTPase enzymes. We found that dUTP photoaffinity labelling was specific for a beta-hairpin motif in HERV-K dUTPase. Mutagenesis of aspartate residues Asp84 and 86 to asparagine within this beta-hairpin showed the carboxylate moiety of both residues was required for catalysis but not for dUTP binding. An increase in the pKa of both aspartate residues brought about by substitution of a serine residue with a glutamate residue adjacent to the aspartate residues increased activity by a factor of 1.67 at pH 8.0, implicating general base catalysis as the enzyme's catalytic mechanism. Conservative mutagenesis of Tyr87 to Phe resulted in a sevenfold reduction of dUTPase activity and a 3.3-fold reduction in binding activity, whilst substitution with an isoleucine residue totally abolished both catalytic activity and dUTP binding, suggesting that binding/activity is dependent on an aromatic side-chain at the base of the hairpin. Comparison of a homology-based three-dimensional model structure of HERV-K dUTPase with a crystallographic structure of the human dUTPase revealed displacement of a conserved alpha-helix in the HERV-K enzyme causing expansion of the HERV-K active site. This expansion may be responsible for the ability of the HERV-K enzyme to hydrolyse dTTP and bind the bulkier dNTPs in contrast to the majority of dUTPases which are highly specific for dUTP. Knowledge of the dUTPase catalytic mechanism and the distinctive topography of the HERV-K active site provides a molecular basis for the design of HERV-K dUTPase-specific inhibitors.

dUTP pyrophosphatase as a potential target for chemotherapeutic drug development.

Aberrant dUTP metabolism plays a critical role in the molecular mechanism of cell killing induced by inhibitors of dihydrofolate reductase and thymidylate synthase. While considerable effort has been directed towards discovering new, more potent inhibitors of these two enzymes, little attention has been given dUTP pyrophosphatase (dUTPase)--the key modulator of cellular dUTP levels--as a potential target for chemotherapeutic drug development. Recent studies have provided evidence that dUTPase is vital for cellular and, in some cases, viral DNA replication. Furthermore, some retroviruses encode dUTPases--a fact which suggests that cellular dUTP metabolism may be more important than previously realized. Here, we briefly review current knowledge of cellular and viral dUTPases and discuss the potential of these enzymes as targets for cancer chemotherapeutic and anti-viral drug development.

A consensus sequence for a functional human endogenous retrovirus K (HERV-K) dUTPase.

Amino acid sequence comparisons have revealed that a potential dUTPase gene is encoded by the retrovirus HERV-K, a defective multicopy virus that is transmitted vertically in humans. This gene is distinct from the human cellular dUTPase gene and thus two potential sources of the enzyme exist in human cells. dUTPases characterized from various sources each contain five conserved amino acid sequence motifs that form the active site of the enzyme. The protein sequence of the putative HERV-K dUTPase deduced from previous DNA sequence data from one proviral clone (HERV-K10) shows marked deviations at highly conserved residues in four of five of these motifs. Therefore, the reported DNA sequence may represent a mutated form of the viral dUTPase gene. To address this possibility, we cloned and sequenced 22 copies of the HERV-K dUTPase gene from human DNA. The results of this analysis indicate that variations evident in the HERV-K10 dUTPase amino acid sequence represent mutations of the wild-type viral DNA sequence. A version of the HERV-K dUTPase gene that corresponds to the ancestral, wild-type DNA sequence was constructed and adapted for expression in Escherichia coli. The resulting enzyme was found to exhibit properties similar to those of dUTPases isolated from other systems. A possible role of the HERV-K dUTPase in human disease is discussed.

Human dUTP pyrophosphatase: uracil recognition by a beta hairpin and active sites formed by three separate subunits.

BACKGROUND: The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP: dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP. RESULTS: The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta strands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirely to main-chain atoms. This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding. Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. CONCLUSIONS: The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture. The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind. These uracil-beta-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein-DNA world. Within the beta hairpin, there is a tyrosine corner motif that normally specifies beta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.

HIV and human endogenous retroviruses: an hypothesis with therapeutic implications.

The enzyme dUTP pyrophosphatase (dUTPase, EC 3.6.1.23) is essential for cellular DNA replication and cell viability by virtue of its role in reducing the availability of dUTP as a substrate for DNA polymerases. Several members of the onco- and lentivirus families of retroviruses encode dUTPases and mutant strains of these viruses defective in this enzyme exhibit suboptimal replication kinetics. Among the lentiviruses there exists a surprising phylogenetic discontinuity in the distribution of dUTPase genes: non-primate viruses (EIAV, CAEV, FIV, visna) contain such genes whereas the primate viruses (HIVs, SIVs) do not. The reason for this difference is unknown. We suggest the following explanation: (1) the nuclear and mitochondrial compartmentalization of the mammalian dUTPase, combined with the cytoplasmic location of ribonucleotide reductase, leads to the net synthesis of dUTP, together with dCTP, dGTP and dATP in the cytoplasm; (2) this combination of dNTPs serves as a "toxic cocktail" for viral replication by virtue of its ability to promote the synthesis of uracil-substituted DNA; (3) many viruses have adapted to this challenge by encoding dUTPases that are free of normal cellular regulatory constraints; and (4) the fortuitous expression of a dUTPase encoded by one or more human endogenous retroviruses (HERVs) has led to the evolutionary loss of the putative ancestral dUTPase gene of primate lentiviruses. Thus, we propose that efficient replication of HIV in humans depends upon expression of a dUTPase encoded by an endogenous retrovirus. If this proposal is correct, then the entry of HIV into target cells is necessary, but not sufficient, for replication of the virus in humans.

MluI site-dependent transcriptional regulation of the Candida albicans dUTPase gene.

The Candida albicans dUTP pyrophosphatase (dUTPase) gene DUT1 has been isolated by genetic complementation in S. cerevisiae. It was found to encode a 17-kDa protein similar in amino-acid sequence to dUTPases isolated from other systems. The gene was adapted for expression in E. coli and yielded a soluble and highly-active enzyme which is easily purified. The 5' flanking sequence of DUT1 contains an MluI site typical of MCB cell-cycle-dependent UAS elements of budding and fission yeast. We found the gene to be cell-cycle-regulated when expressed in S. cerevisiae, and deletion of the MluI site resulted in a large reduction of DUT1 transcription in C. albicans. These results suggest that MCB elements are functionally conserved in this pathogenic fungus. Based on the vital role that dUTPase plays in DNA replication, the C. albicans enzyme may be a potentially useful target for the development of novel anti-fungal compounds.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

Cloning and molecular characterization of three genes, including two genes encoding serine hydroxymethyltransferases, whose inactivation is required to render yeast auxotrophic for glycine.

The genes encoding both the cytosolic and mitochondrial serine hydroxymethyltransferases (SHM2 and SHM1, respectively) and a third unidentified gene of the yeast Saccharomyces cerevisiae have been isolated and their nucleotide sequences determined. Analysis of the predicted amino acid sequence of the amino-terminal regions, sequence comparison with other genes encoding SHMT enzymes, and subcellular fractionation studies all suggested that the SHM1 gene encodes the mitochondrial SHMT, while the SHM2 gene encodes the cytosolic enzyme. The SHM2 gene but not the SHM1 gene has putative GCN4 sites upstream of the putative TATA box, suggesting regulation of its transcription by the general amino acid control system. Yeast mutants with disruptions at each SHM gene and in both genes were constructed and all mutants had the same growth requirements as the parental strains. Mutagenesis of the double-disrupted, shm1 shm2 yeast yielded strains of a single complementation group that are auxotrophic for glycine. Complementation of the glycine auxotrophy using a yeast genomic library retrieved the SHM1 and SHM2 genes and a third gene designated GLY1. Gene disruption studies demonstrated that inactivation of SHM1, SHM2, and GLY1 is required to yield yeast that are completely auxotrophic for glycine.

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.

Expression of DNA replication genes in the yeast cell cycle.

In recent years, numerous studies using a wide variety of systems have clearly established some of the fundamental components of eukaryotic cell-division control. These include p34cdc2 protein kinases (henceforth referred to as p34) and closely related proteins (p33cdc2), and the members of the cyclin gene family which, through interaction with the p34 (and p33) kinases, regulate transitions from one stage of the cell cycle to the next. The function of these proteins in the cell cycle has been conserved to the extent that p34 protein kinase and cyclin genes are, in some cases, interchangeable between organisms. Despite the tremendous insight that studies on p34 and the cyclins have provided, many questions remain about the details of the molecular events which allow these proteins to govern cell division. One question of particular interest concerns the means by which p34 interaction with G1 phase cyclins promotes G1 to S phase transition in the cell cycle. This is of primary importance since entry into the cell cycle is regulated, for most cells, by passage from G1 (or G0) into S phase. Recent findings in the yeast Saccharomyces cerevisiae point to a potential link between the p34/G1 cyclin protein kinase complex and the regulation of DNA replication genes during the cell cycle. This paper reviews studies dealing with the transcriptional control of DNA replication genes in yeast and also briefly discusses the potential role of G1 cyclins in this process. A similar review of this subject has also been given by Johnston and Lowndes (1992).

MCB elements and the regulation of DNA replication genes in yeast.

In eukaryotic organisms, genes involved in DNA replication are often subject to some form of cell cycle control. In the yeast Saccharomyces cerevisiae, most of the DNA replication genes that have been characterized to date are regulated at the transcriptional level during G1 to S phase transition. A cis-acting element termed the MluI cell cycle box (or MCB) conveys this pattern of regulation and is common among more than 20 genes involved in DNA synthesis and repair. Recent findings indicate that the MCB element is well conserved among fungi and may play a role in controlling entry into the cell division cycle. It is evident from studies in higher systems, however, that transcriptional regulation is not the only form of control that governs the cell-cycle-dependent expression of DNA replication genes. Moreover, it is unclear why this general pattern of regulation exists for so many of these genes in various eukaryotic systems. This review summarizes recent studies of the MCB element in yeast and briefly discusses the purpose of regulating DNA replication genes in the eukaryotic cell cycle.

Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.

Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.

Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate.

Disruption of the dCMP deaminase (DCD1) gene, or provision of excess dTMP to a nucleotide-permeable strain, produced dramatic increases in the dCTP or dTTP pools, respectively, in growing cells of the yeast Saccharomyces cerevisiae. The mutation rate of the SUP4-o gene was enhanced 2-fold by the dCTP imbalance and 104-fold by the dTTP imbalance. 407 SUP4-o mutations that arose under these conditions, and 334 spontaneous mutations recovered in an isogenic strain having balanced DNA precursor levels, were characterized by DNA sequencing and the resulting mutational spectra were compared. Significantly more (greater than 98%) of the changes resulting from nucleotide pool imbalance were single base-pair events, the majority of which could have been due to misinsertion of the nucleotides present in excess. Unexpectedly, expanding the dCTP pool did not increase the fraction of A.T----G.C transitions relative to the spontaneous value nor did enlarging the dTTP pool enhance the proportion of G.C----A.T transitions. Instead, the elevated levels of dCTP or dTTP were associated primarily with increases in the fractions of G.C----C.G or A.T----T.A. transversions, respectively. Furthermore, T----C, and possibly A----C, events occurred preferentially in the dcd1 strain at sites where dCTP was to be inserted next. C----T and A----T events were induced most often by dTMP treatment at sites where the next correct nucleotide was dTTP or dGTP (dGTP levels were also elevated by dTMP treatment). Finally, misinsertion of dCTP or dTTP did not exhibit a strand bias. Collectively, our data suggest that increased levels of dCTP and dTTP induced mutations in yeast via nucleotide misinsertion and inhibition of proofreading but indicate that other factors must also be involved. We consider several possibilities, including potential roles for the regulation and specificity of proofreading and for mismatch correction.

Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae.

Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMP1. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMP1. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMP1 MluI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.