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471.
M. Salas R. Freire M.S. Soengas J.A. Esteban J. Méndez A. Bravo M. Serrano M.A. Blasco J.M. Lázaro L. Blanco C. Gutiérrez J.M. Hermoso 《FEMS microbiology reviews》1995,17(1-2):73-82
Abstract: φ29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3'–5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions. 相似文献
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By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication. 相似文献
474.
J. L. Carrasco A. Chueca F. E. Prado R. Hermoso J. J. Lázaro J. L. Ramos M. Sahrawy J. López Gorgé 《Planta》1994,193(4):494-501
A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library
in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for
the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a
3′-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular
mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase
(kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for
the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears
to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol
plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry.
In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual
activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results
show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different
expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric
structure of the native chloroplast enzyme.
Accession number for pea chloroplast FBPase coding sequence: X68826 in the European Molecular Biology Laboratory (EMBL) 相似文献
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