1 Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique, Université de Lyon, 7 passage du Vercors, 69367 Lyon Cedex 07, France
2 National Institute for Medical Research, Division of Mycobacterial Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Correspondence
Alain J. Cozzone
aj.cozzone{at}ibcp.fr
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ABSTRACT |
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INTRODUCTION |
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Protein phosphorylation catalysed by protein kinases is a frequent post-translational modification used by organisms to transduce extracellular signals into cellular functions. Its occurrence was first demonstrated in eukaryotes, then later in prokaryotes, and it is now considered a universal modification of proteins allowing all living systems to respond to environmental variations (Hunter, 1995). In the case of M. tuberculosis, the presence of proteins possessing phosphorylated tyrosine residues has been detected using an antiphosphotyrosine antibody, but none of them has been identified (Chow et al., 1994
). The analysis of the total genome sequence of the bacterium has suggested the presence of eleven putative protein kinases and four phosphoprotein phosphatases (Cole et al., 1998
). Recently, two protein tyrosine phosphatases (Koul et al., 2000
) and five serine/threonine kinases, PknD (Peirs et al., 1997
), PknB (Av-Gay et al., 1999
), PknF, PknG (Koul et al., 2001
) and PknA (Chaba et al., 2002
), have been cloned and characterized. However, the biological function of these enzymes has not been determined, with the exception of PknA, which might have a regulatory role in cell division (Chaba et al., 2002
).
As Hsp 16·3 and Hsp 70 are major antigens in mycobacterial infections and because of the presence of protein phosphorylating activities in M. tuberculosis, we examined the possibility that these proteins could undergo phosphorylation, and thus whether such modifications should be taken into account when deciphering the molecular pathogenesis of tuberculosis.
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METHODS |
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Culture media and growth conditions.
E. coli strains were grown in LB or 2YT medium at 37 °C. For strains carrying drug-resistance genes, ampicillin was added to the medium at 50 µg ml1. Cultures of M. tuberculosis were grown in Dubos medium at 37 °C. Growth was monitored by measuring OD600. The cells were collected in stationary phase.
Preparation of total cellular extracts.
For preparing cellular extracts of M. tuberculosis, bacteria were first collected by centrifugation. The pellet was washed in 20 mM MOPS at pH 7·5, 5 mM NaCl, 1 mM EDTA and 1 mM DTT, and cells were resuspended in the same buffer, then subjected to mechanical bead breakage for 2x25 s. To remove cell debris, extracts were centrifuged at 14 000 g for 30 min. The resulting supernatant was filtered and glycerol was added to a final concentration of 10 % (v/v). Cell lysates were split into aliquots and frozen at 70 °C.
Two-dimensional separation of proteins.
Proteins (about 200 µg) from a whole-cell extract of M. tuberculosis were analysed using the O'Farrell gel technique (O'Farrell et al., 1977). Separation in the first dimension was achieved by non-equilibrium isoelectric focussing in 4 % acrylamide and ampholines in the pH range of 310 in the presence of 9·5 M urea. Separation in the second dimension was performed by SDS-PAGE in 12·5 % polyacrylamide. After protein separation, gels were soaked in 16 % trichloroacetic acid and heated for 10 min at 95 °C. Proteins were then visualized by silver staining, and phosphoproteins were detected by autoradiography. Radioactive spots were excised from the gel and analysed by mass spectrometry.
Identification of proteins by mass spectrometry.
Protein spots of interest were cut from the gel using a pipette tip, placed in 0·5 ml microcentrifuge tubes, and processed as described by Courchesne & Patterson (1999) with some modifications. The gel was washed once with MilliQ water and twice with 25 mM NH4HCO3, then destained and dehydrated by washing three times (
5 min each) with a solution containing equal volumes of 50 mM NH4HCO3 and acetonitrile. The destained gel pieces were rehydrated twice with 50 mM NH4HCO3 and proteins were reduced by incubation with 20 mM DTT in 50 mM NH4HCO3 for 1 h at 37 °C, then washed again briefly with 50 mM NH4HCO3. The proteins were alkylated with 25 mM iodoacetamide in 50 mM NH4HCO3 and incubated at room temperature in the dark for 30 min, washed twice with 50 mM NH4HCO3, and dehydrated three times with acetonitrile (
5 min each). The gel was left to dry at room temperature and stored at 70 °C. The protein was digested overnight at 32 °C with a minimal amount (
10 µl) of ice-cold 5 mM NH4HCO3 containing 2 µg trypsin µl1. The peptide samples were acidified by addition of 0·1 vol. 2 % trifluoroacetic acid prior to mass spectrometry. Thin layer matrix surfaces of
-cyano-4-hydroxycinnamic acid mixed with nitrocellulose were prepared as described by Shevchenko et al. (1996)
. The acidified digest was loaded onto the thin layer and allowed to dry prior to rinsing with water. A Reflex III matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonik) equipped with a Scout-384 probe was used to obtain positive ion mass spectra of digested proteins. Peptide mass fingerprints were searched against the non-redundant database (available from the National Center for Biotechnology Information). Partial enzymic cleavage sites, oxidation of methonine, pyroglutamic acid formation at N-terminal glutamine and modification of cysteine by acrylamide were considered in these searches.
Isolation of chromosomal DNA from M. tuberculosis.
One colony of M. tuberculosis was inoculated into 10 ml Dubos medium containing the appropriate supplements and incubated at 37 °C. After growth for 12 weeks, bacteria were subcultured into 150 ml Dubos medium. The culture was grown to an OD600 of 0·71·0, and 0·1 vol. 2 M glycine was added 20 h before harvest. Bacteria were pelleted by centrifugation at 16 000 g for 20 min at 4 °C and the cell pellets were resuspended in 2 ml TE (10 mM Tris/HCl, pH 7·5, 1 mM EDTA) then divided into 500 µl aliquots. The tubes containing the cell suspensions were incubated at 80 °C for 1 h to kill the bacteria. A mixture containing 50 µl lysozyme and lipase (2 mg ml1 each in TE) and 5 µl DNase-free RNase (0·5 µg µl1) was added to each suspension and incubated at 37 °C for 2 h. Tubes were then frozen in an ethanol/dry ice bath for 10 min followed by incubation at 75 °C for 10 min. After cooling to room temperature, proteinase K and SDS were added to final concentrations of 500 µg ml1 and 0·5 % (w/v), respectively, and the mixture incubated at 50 °C for 1 h. The solution was extracted twice with 0·5 ml phenol/chloroform/isoamyl alcohol (25/24/1, by vol.) and once with 0·5 ml chloroform. Chromosomal DNA present in the aqueous phase was precipitated by the addition of 0·02 vol. 5 M NaCl and 2 vols 99 % ethanol at room temperature. The chromosomal DNA was collected by centrifugation at 11 600 g for 10 min at 4 °C, then washed twice with 1 ml 70 % ethanol. After removing traces of ethanol by brief centrifugation, the DNA pellet was air-dried at room temperature, dissolved in 300 µl TE and stored at 4 °C. The amount of DNA was determined by measuring A260 in a Thermo Unicam UV2 spectrophotometer, and chromosomal DNA was analysed by agarose gel electrophoresis.
Expression and purification of Hsp 16·3.
The hsp16·3 gene was amplified, with appropriate restriction endonuclease cleavage sites at both ends, from genomic DNA from M. tuberculosis H37Rv using the primers 5'-GGA ATT CCA TAT GGC CAC CAC CCT TCC CGT TC-3' and 5'-CGC GGA TCC TCA GTT GGT GGA CCG GAT CTG-3'. The amplified fragment was subcloned into the pET15 expression vector to yield the plasmid pET-hsp16·3. The nucleotide sequence of the amplified gene was checked by dideoxynucleotide sequencing (Sanger et al., 1977). The pET-hsp16·3 expression plasmid was used to transform competent E. coli BL21(DE3) cells. Overproduction of the 6His-Hsp16·3 protein was obtained by induction with 1 mM IPTG. After 2 h, 6His-Hsp 16·3 was extracted and purified using an immobilized Zn2+ matrix, suitable for purification of fusion proteins carrying a polyhistidine tag. Production of the 6His-Hsp 16·3 protein was confirmed by analysis of Coomassie-blue-stained polyacrylamide gels. Protein concentration was determined using the Coomassie Plus Protein Assay (Pierce).
In vitro phosphorylation assay.
In vitro phosphorylation of about 5 µg purified 6His-Hsp 16·3 protein or Hsp 70 protein (Sigma) was performed for 10 min at 37 °C in 20 µl of a buffer containing 25 mM Tris/HCl (pH 7·0), 1 mM DTT, 1 mM EDTA, 5 mM MgCl2 and 200 µCi (7·4 MBq) [-32P]ATP ml1. In some assays, MgCl2 was replaced with 5 mM MnCl2, ZnCl2 or CaCl2. In each case, the reaction was stopped by addition of an equal volume of 2x sample buffer (Laemmli, 1970
). After electrophoresis, gels were soaked in 16 % trichloroacetic acid for 10 min at 90 °C. They were stained with Coomassie blue and radioactive proteins were visualized by autoradiography.
Analysis of the phosphoamino acid content of proteins.
Protein samples were separated by one-dimensional gel electrophoresis (Laemmli, 1970), then electroblotted onto an Immobilon PVDF membrane. Phosphorylated proteins bound to the membrane were detected by autoradiography. The 32P-labelled protein bands were excised from the Immobilon blot and hydrolysed in 6 M HCl for 1 h at 110 °C. The acid-stable phosphoamino acids thus liberated were separated by electrophoresis in the first dimension at pH 1·9 (800 V h) in 7·8 % acetic acid/2·5 % formic acid, followed by ascending chromatography in the second dimension in 2-methyl-1-propanol/formic acid/water (8/3/4, by vol.). After migration, radioactive molecules were detected by autoradiography. Authentic phosphoserine, phosphothreonine and phosphotyrosine were run in parallel and visualized by staining with ninhydrin (Duclos et al., 1991
).
ATP binding.
Photoaffinity [-32P]ATP cross-linking experiments were performed as described by Matsuyama et al. (1990)
. The 6His-Hsp 16·3 protein was incubated in a buffer containing 25 mM Tris/HCl, pH 7·0, 1 mM DTT, 1 mM EDTA, 5 mM MgCl2 and [
-32P]ATP (3000 Ci mmol1; 110 TBq mmol1) at 37 °C for 10 min. The reaction was stopped by placing the mixture on ice. The mixture was then subjected to affinity cross-linking for 45 min at 4 °C using a 254 nm lamp at a distance of 4 cm. Cross-linking reactions were carried out with or without UV irradiation in the presence or absence of ATP at various concentrations. The reaction was stopped by addition of 2x Laemmli buffer. After boiling, samples were subjected to SDS-PAGE in 10 % polyacrylamide gels and radioactive bands were visualized by autoradiography.
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RESULTS |
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DISCUSSION |
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Autophosphorylation has been demonstrated previously in other heat-shock proteins from mycobacteria, including DnaK and GroEL (McCarty & Walker, 1991; Nadeau et al., 1993
; Zylicz et al., 1983
). This observation was confirmed in our study, which found Hsp 70 can autophosphorylate and thus exhibits functional properties characteristic of the Hsp 70 family. Such functional similarity is further demonstrated by the stimulatory effect of calcium ions on the phosphorylation activity of Hsp 70. The autokinase activity of Hsp 16·3 and Hsp 70 reported here adds to the biochemical similarities between the large and small Hsp families, and suggests that they may share common mechanisms in their mode of action, even though the amino acid residues phosphorylated in Hsp 16·3 (serine) are different from those modified in Hsp 70 (threonine).
Besides these autokinases, other protein kinase activities have been described in mycobacteria. Mycobacterium smegmatis has been reported to contain a soluble calcium/calmodulin-dependent 35 kDa protein kinase with a narrow substrate specificity for both exogenous and endogenous substrates (Sharma et al., 1998). Similarly, M. tuberculosis contains eleven eukaryotic-like serine/threonine protein kinases, the Pkn family, some of which have been characterized in detail (Chaba et al., 2002
; Koul et al., 2001
). The presence of three phosphoproteins of 50, 55 and 60 kDa, all phosphorylated at tyrosine residues in M. tuberculosis, has also been described, although the enzymic activity responsible for the phosphorylation has not yet been characterized (Chow et al., 1994
). Two-component phosphorylation systems are also found in mycobacteria. Several examples of such systems have been reported in M. tuberculosis (Haydel et al., 1999
; Mayuri et al., 2002
), as well as in M. smegmatis (O'Toole et al., 2003
). Together these data indicate that protein phosphorylation is likely to play a crucial role in the metabolism and physiology of mycobacteria (Drews et al., 2001
). Blockage of protein kinase activity with specific inhibitors has already been shown to result in limitation of growth (Drews et al., 2001
) and a reduction in phagocytosis by macrophages (Prabhakaran et al., 2000
).
The phosphoproteins of the Hsp 70 family have been reported to participate in various cellular functions, including binding to unfolded or nascent polypeptides and the renaturation and disaggregation of misfolded polypeptides. Their phosphorylation is known to induce an increased affinity for polypeptide substrates (Peake et al., 1998). These proteins are also involved in replication and protein transport within the cell, and the extent of their phosphorylation is considered an indicator of the level of stress imposed by environmental conditions (Hengge & Bukau, 2003
).
In contrast, little is known of the biological role of the phosphorylation of Hsp 16·3. Hsp 16·3 appears to consist of a trimer of trimers with a total molecular mass of 149 kDa (Chang et al., 1996). There may be a relationship between the aggregation of this protein complex and the extent of its phosphorylation. This is seen in several heat-shock proteins, including the
-crystallin protein homologous to Hsp 16·3, in which the disaggregation of the oligomeric structure is accompanied by enhanced phosphorylation (Kato et al., 1994
). However, no such relationship was observed after treatment of Hsp 16·3 with the detergent sodium deoxycholate (data not shown).
Hsp 16·3, which is an immunodominant antigen overproduced during the latent stationary phase of M. tuberculosis infection, also seems to act as a chaperone through an ATP-independent process (Groenen et al., 1994; Horwitz, 1992
). However, our results from UV cross-linking experiments show that ATP tightly interacts with Hsp 16·3, although it cannot be determined whether the ATP molecules involved in this interaction are those which serve as donors in the phosphorylation reaction. Our results are consistent with a recent report indicating that Hsp 16·3 from M. tuberculosis was protected by ATP against proteolysis by chymotrypsin, whereas no effect was found with a nonhydrolysable analogue of ATP (Valdez et al., 2002
), suggesting a close interaction between ATP and Hsp 16·3. However, Hsp 16·3 may bind to other nucleotides, and further studies are needed to evaluate the specificity of the Hsp 16·3/ATP interaction.
The kinase activity of Hsp 16·3 may not be restricted to its own phosphorylation. It may also act on other protein substrates, either within the mycobacterial cell or within the infected cells. It would be interesting to investigate whether there is a phosphoprotein phosphatase activity that can dephosphorylate Hsp 16·3 and/or other substrates. The extent of phosphorylation of Hsp 16·3 should be measured under various physiological conditions in order to evaluate the effect of the environment on modification of this protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chaba, R., Raje, M. & Chakraborti, P. K. (2002). Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division. Eur J Biochem 269, 10781085.
Chang, Z., Primm, T. P., Jakana, J., Lee, I. H., Serysheva, I., Chiu, W., Gilbert, H. F. & Quiocho, F. A. (1996). Mycobacterium tuberculosis 16-kDa antigen (Hsp16·3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J Biol Chem 271, 72187223.
Chow, K., Ng, D., Stokes, R. & Johnson, P. (1994). Protein tyrosine phosphorylation in Mycobacterium tuberculosis. FEMS Microbiol Lett 124, 203207.[CrossRef][Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Courchesne, P. L. & Patterson, S. D. (1999). Identification of proteins by matrix-assisted laser desorption/ionization mass spectrometry using peptide and fragment ion masses. Methods Mol Biol 112, 487511.[Medline]
Cozzone, A. J. (1993). ATP-dependent protein kinases in bacteria. J Cell Biochem 51, 713.[Medline]
Dannenberg, A. M., Jr (1993). Immunopathogenesis of pulmonary tuberculosis. Hosp Pract (Off Ed) 28, 5158.
Drews, S. J., Hung, F. & Av-Gay, Y. (2001). A protein kinase inhibitor as an antimycobacterial agent. FEMS Microbiol Lett 205, 369374.[CrossRef][Medline]
Duclos, B., Marcandier, S. & Cozzone, A. J. (1991). Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol 201, 1021.[Medline]
Groenen, P. J., Merck, K. B., de Jong, W. W. & Bloemendal, H. (1994). Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology. Eur J Biochem 225, 119.[Abstract]
Haydel, S. E., Dunlap, N. E. & Benjamin, W. H., Jr (1999). In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TrcR/TrcS proteins. Microb Pathog 26, 195206.[CrossRef][Medline]
Hengge, R. & Bukau, B. (2003). Proteolysis in prokaryotes: protein quality control and regulatory principles. Mol Microbiol 49, 14511462.[CrossRef][Medline]
Horwitz, J. (1992). Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 89, 1044910453.[Abstract]
Hunter, T. (1995). Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225236.[Medline]
Kato, K., Hasegawa, K., Goto, S. & Inaguma, Y. (1994). Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol Chem 269, 1127411278.
Koul, A., Choidas, A., Treder, M., Tyagi, A. K., Drlica, K., Singh, Y. & Ullrich, A. (2000). Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. J Bacteriol 182, 54255432.
Koul, A., Choidas, A., Tyagi, A. K., Drlica, K., Singh, Y. & Ullrich, A. (2001). Serine/threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization. Microbiology 147, 23072314.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Matsuyama, S., Kimura, E. & Mizushima, S. (1990). Complementation of two overlapping fragments of SecA, a protein translocation ATPase of Escherichia coli, allows ATP binding to its amino-terminal region. J Biol Chem 265, 87608765.
Mayuri, Bagchi, G., Das, T. K. & Tyagi, J. S. (2002). Molecular analysis of the dormancy response in Mycobacterium smegmatis: expression analysis of genes encoding the DevR-DevS two-component system, Rv3134c and chaperone alpha-crystallin homologues. FEMS Microbiol Lett 211, 231237.[CrossRef][Medline]
McCarty, J. S. & Walker, G. C. (1991). DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A 88, 95139517.[Abstract]
Moreno, C., Mehlert, A. & Lamb, J. (1988). The inhibitory effects of mycobacterial lipoarabinomannan and polysaccharides upon polyclonal and monoclonal human T cell proliferation. Clin Exp Immunol 74, 206210.[Medline]
Nadeau, K., Das, A. & Walsh, C. T. (1993). Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J Biol Chem 268, 14791487.
O'Farrell, P. Z., Goodman, H. M. & O'Farrell, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 11331141.[Medline]
Olsen, A. W. & Andersen, P. (2003). A novel TB vaccine; strategies to combat a complex pathogen. Immunol Lett 85, 207211.[CrossRef][Medline]
O'Toole, R., Smeulders, M. J., Blokpoel, M. C., Kay, E. J., Lougheed, K. & Williams, H. D. (2003). A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis. J Bacteriol 185, 15431554.
Peake, P., Winter, N. & Britton, W. (1998). Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics. Biochim Biophys Acta 1387, 387394.[Medline]
Peirs, P., De Wit, L., Braibant, M., Huygen, K. & Content, J. (1997). A serine/threonine protein kinase from Mycobacterium tuberculosis. Eur J Biochem 244, 604612.[Abstract]
Prabhakaran, K., Harris, E. B. & Randhawa, B. (2000). Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. J Med Microbiol 49, 339342.
Roche, P. W., Peake, P. W., Davenport, M. P. & Britton, W. J. (1994). Identification of a Mycobacterium leprae-specific T cell epitope on the 70 kDa heat shock protein. Immunol Cell Biol 72, 215221.[Medline]
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 54635467.[Abstract]
Sharma, S., Giri, S. & Khuller, G. K. (1998). Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607. Mol Cell Biochem 183, 183191.[CrossRef][Medline]
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins on silver-stained polyacrylamide gels. Anal Chem 68, 850858.[CrossRef][Medline]
Sudre, P., ten Dam, G. & Kochi, A. (1992). Tuberculosis: a global overview of the situation today. Bull World Health Organ 70, 149159.[Medline]
Valdez, M. M., Clark, J. I., Wu, G. J. & Muchowski, P. J. (2002). Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16·3 and human alphaB-crystallin. Eur J Biochem 269, 18061813.
Verbon, A., Hartskeerl, R. A., Schuitema, A., Kolk, A. H., Young, D. B. & Lathigra, R. (1992). The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins. J Bacteriol 174, 13521359.[Abstract]
Wayne, L. G. (1994). Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13, 908914.[Medline]
Yuan, Y., Crane, D. D. & Barry, C. E., 3rd (1996). Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog. J Bacteriol 178, 44844492.[Abstract]
Zylicz, M., LeBowitz, J. H., McMacken, R. & Georgopoulos, C. (1983). The DnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc Natl Acad Sci U S A 80, 64316435.[Abstract]
Received 26 September 2003;
revised 16 April 2004;
accepted 27 April 2004.
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