A bacterial calcium-binding protein homologous to calmodulin

Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand. Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium. However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins. We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene. It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin. This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.     

The structure of the E. coli recA protein monomer and polymer.

The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.     

A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin

Infection of tobacco plants with tobacco mosaic virus (TMV) results in an increase in the activities of several enzymes and induces the de novo synthesis of about 10 proteins that are protease-resistant and soluble at pH 3. These proteins accumulate in the intracellular leaf space. The appearance of pathogenesis-related (PR) proteins is closely associated with the phenomenon of 'systemic acquired resistance' and it has been suggested that such proteins have an antiviral function. Previously, we cloned complementary DNAs to the messenger RNAs for the three smallest PR proteins, PR-1a, -1b and -1c, and these clones were used to show that there is an increase of more than 100-fold in the concentration of PR-1 mRNAs following TMV infection of tobacco. Here, we describe the cDNA cloning of another mRNA whose synthesis is induced by TMV infection. Sequencing of the cDNA showed that the encoded protein is highly homologous to thaumatin, the intensely sweet-tasting protein from the fruits of the monocot Thaumatococcus daniellii Benth, a West African rainforest shrub. The conservation of a gene encoding a thaumatin-like protein in tobacco suggests that the encoded protein may have a more general function than that of being sweet-tasting.     

Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF.

A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter. Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62. Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE. Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE. Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins. Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80. But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1. The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF. Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage.

Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.     

Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination.

The repair of DNA double-strand breaks is essential for cells to maintain their genomic integrity. Two major mechanisms are responsible for repairing these breaks in mammalian cells, non-homologous end-joining (NHEJ) and homologous recombination (HR): the importance of the former in mammalian cells is well established, whereas the role of the latter is just emerging. Homologous recombination is presumably promoted by an evolutionarily conserved group of genes termed the Rad52 epistasis group. An essential component of the HR pathway is the strand-exchange protein, known as RecA in bacteria or Rad51 in yeast. Several mammalian genes have been implicated in repair by homologous recombination on the basis of their sequence homology to yeast Rad51: one of these is human XRCC2. Here we show that XRCC2 is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids. We find that hamster cells deficient in XRCC2 show more than a 100-fold decrease in HR induced by double-strand breaks compared with the parental cell line. This defect is corrected to almost wild-type levels by transient transfection with a plasmid expressing XRCC2. The repair defect in XRCC2 mutant cells appears to be restricted to recombinational repair because NHEJ is normal. We conclude that XRCC2 is involved in the repair of DNA double-strand breaks by homologous recombination.     

MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.

Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product

Organisms amenable to easy genetic analysis should prove helpful in assessing the function of at least those proto-oncogene products which are highly conserved in different eukaryotic cells. One obvious possibility is to pursue the matter in Drosophila melanogaster DNA, which has sequences homologous to several vertebrate oncogenes. Another is to turn to the yeast Saccharomyces cerevisiae, if it contains proto-oncogene sequences. Here we report the identification of a gene in S. cerevisiae which codes for a 206 amino acid protein (YP2) that exhibits striking homology to the p21 products of the human c-has/bas proto-oncogenes and the transforming p21 proteins of the Harvey (v-rasH) and Kirsten (v-rasK) murine sarcoma viral oncogenes. The YP2 gene is located between the actin and the tubulin gene on chromosome VI and is expressed in growing cells. The protein it encodes might share the nucleotide-binding capacity of p21 proteins.     

An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2

Recently the human T cell erythrocyte receptor CD2 has been shown to bind human erythrocytes through LFA-3, a heavily glycosylated surface protein of broad tissue distribution. CD2-LFA-3 interactions are important for cytolytic conjugate formation, for thymocyte adhesion, and for T cell activation. A complementary DNA clone encoding LFA-3 was isolated using a complementary DNA clone encoding LFA-3 was isolated using a novel transient expression system of mouse cells. The cDNA encodes a phospholipid-linked membrane protein whose extracellular domain shares significant homology with CD2. As CD2 is homologous with the neural cell adhesion molecule NCAM in immunoglobulin-like domains, cellular adhesion molecules in both neural and lymphoid tissues could have a common ancestor.     

Electron transfer from protein to DNA in irradiated chromatin

Irradiation of dry or fully hydrated frozen DNA systems (conditions of direct damage) has been shown by electron-spin resonance spectroscopy to give rise to electron-gain centres localized on thymine (T.-) and electron loss centres ('holes') localized on guanine (G.+) with approximately equal yields. Our parallel studies on the development of both single- and double-strand breaks under comparable conditions provide good evidence that these radical centres are the precursors to such damage, and we and others have argued that this may be of relevance to the damage pathways in vivo. We now report evidence that when DNA is complexed to proteins as it is in the nuclei of eukaryotes, electron transfer from the histone to DNA is facile, leading to a significant increase in the yield of electron-gain centres in DNA as judged from their electron-spin resonance spectra. In contrast 'holes' generated in the protein are trapped and do not lead to any detectable increase in the yields of G.+.     

The Cdt1 protein is required to license DNA for replication in fission yeast

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.