Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway

Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells. From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced. The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction. HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes. Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein. HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase.     

A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA. A role for helicase motif III

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.     

The yeast mic2 mutant is defective in the formation of mannosyl-diinositolphosphorylceramide

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243-246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10-11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2-3 [Yamaguchi et al., Biochemistry 31 (1992) 8344-8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.     

KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion

The KARP-1 (Ku86 Autoantigen Related Protein-1) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.     

Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735-12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol. Chem. 258 (1983) 12632-12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293-1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141-144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15-21 [21]]. The alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559-601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II alpha- or beta-isoform, and of mutant alpha-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant alpha-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759-16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59-70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca2+-independent activity of calmodulin-dependent protein kinase II alpha subunit, J. Biochem. 109 (1991) 137-143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559-567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells-H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129-141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the beta than alpha cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. (c) 1998 Elsevier Science B.V. All rights reserved.     

The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation

The S. cerevisiae SIS1 gene is essential and encodes a heat shock protein with similarity to the bacterial DnaJ protein. At the nonpermissive temperature, temperature-sensitive sis1 strains rapidly accumulate 80S ribosomes and have decreased amounts of polysomes. Certain alterations in 60S ribosomal subunits can suppress the temperature-sensitive phenotype of sis1 strains and prevent the accumulation of 80S ribosomes and the loss of polysomes normally seen under conditions of reduced SIS1 function. Analysis of sucrose gradients for SIS1 protein shows that a large fraction of SIS1 is associated with 40S ribosomal subunits and the smaller polysomes. These and other results indicate that SIS1 is required for the normal initiation of translation. Because DnaJ has been shown to mediate the dissociation of several protein complexes, the requirement of SIS1 in the initiation of translation might be for mediating the dissociation of a specific protein complex of the translation machinery.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

A yeast homologue of the human phosphotyrosyl phosphatase activator PTPA is implicated in protection against oxidative DNA damage induced by the model carcinogen 4-nitroquinoline 1-oxide

The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties. However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species. To elucidate the response of eukaryotic cells to 4-NQO-induced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen. One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation. A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated. Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate. Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair. These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.     

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases

The lung is constantly exposed to invading particulate matter and potential pathogens. To cope with this pressure, the lung has evolved a sophisticated, multitiered defense mechanism designed to clear offending agents while inducing a minimum amount of concomitant inflammation. Mechanical defense mechanisms first attempt to remove material physically from the tracheobronchial tree. Particulate matter and pathogens that circumvent this first line of defense are ingested by resident and recruited phagocytes in the lower respiratory tract and alveoli. If phagocytic defenses are impaired or overwhelmed, specific immune mechanisms become operational and lead to the generation of delayed-type hypersensitivity (granulomatous), cytotoxic, and humoral (antibody) responses. Congenital or acquired impairment of pulmonary host defenses can occur at any of these steps. Impairment of a particular component of pulmonary host defense is usually associated with a characteristic spectrum of infectious and noninfectious pulmonary complications. Thus, understanding all the components of pulmonary host defense and how to evaluate them will greatly aid the physician who cares for immunocompromised patients with lung disease.     

Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing

In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.     

Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.     

Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast

A checkpoint responding to DNA damage in G2 results in a delay in the onset of mitosis through inhibition of p34cdc2 kinase activity via maintenance of inhibitory tyrosine phosphorylation. Genetic analyses of this checkpoint in fission yeast have identified single alleles of several genes, suggesting these screens are not yet saturating, and hence further genes await identification. To fully understand the complexity of this checkpoint it will be necessary to define all the genes involved. To this end we screened for new mutants defective in the ability to delay mitosis in the presence of DNA-damaging agents. Twenty-four mutants were isolated that were defective in UV-C and MMS-induced checkpoint delay. Amongst these mutants was an allele of cut5 that was also defective in the checkpoint responses. We show here, contrary to previous reports, that the UV-C induced checkpoint response is defective in cut5 mutants. Therefore, like all other checkpoint mutants, cut5 is required for G2 checkpoint arrest following DNA damage, regardless of the nature of the lesions involved.     

Discrete start sites for DNA synthesis in the yeast ARS1 origin

Sites of DNA synthesis initiation have been detected at the nucleotide level in a yeast origin of bidirectional replication with the use of replication initiation point mapping. The ARS1 origin of Saccharomyces cerevisiae showed a transition from discontinuous to continuous DNA synthesis in an 18-base pair region (nucleotides 828 to 845) from within element B1 toward B2, adjacent to the binding site for the origin recognition complex, the putative initiator protein.     

DNA unwinding in the CYC1 and DED1 yeast promoters

The superiority of intensive versus standard chemotherapy for aggressive (I: intermediate; H: high grade) NHL is still debated; increased antitumor activity may be counterbalanced by increased toxicity. We have designed a first-line five-drug regimen (vincristine, idarubicin, cyclophosphamide, etoposide and deflazacort), with the aim of potentiating the CHOP protocol without losing tolerability and ease of administration. Seventy-one patients (33% aged > or = 65) entered the study. CR was obtained in 66.7% of patients (I: 74%; H: 56%), PR in 19.7%: overall response rate was 86.4%. Six patients were resistant, two died during treatment. With a median follow up of two years, relapse has occurred in 14 patients (8 I, 6 H). At 3 years, overall survival was projected to be 62.5% (I 73.5%; H 31.4%), disease free survival 66% (I 71%, H 56.3%). No organ toxicity occurred. Myelosuppression was moderate, with a nadir on the 14th day. Febrile episodes occurred in 16% of courses, dose delay in 19% of courses; dose reduction in 3% of patients. No patient required hospitalization. G-CSF was only occasionally used. This regimen has shown a potent antitumor effect with an excellent tolerance, even in elderly patients.     

Characterization of yeast sar1 temperature-sensitive mutants, which are defective in protein transport from the endoplasmic reticulum

SAR1 encodes a low molecular weight GTPase that is essential in the early process of vesicular transport in the secretory pathway. By random and site-directed mutagenesis of the SAR1 gene, we have obtained three temperature-sensitive mutants, N132I, E112K, and D32G. They all show a defect in transport from the endoplasmic reticulum to the Golgi apparatus, and accumulate endoplasmic reticulum membranes at the restrictive temperature. This is consistent with our previous observations in vivo on a galactose-shutoff mutant as well as the in vitro results, and provides powerful tools for further genetic analyses.