Cloning, sequencing and biochemical characterization of RecA and RuvB from divergent thermophilic bacteria.
Item
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Title
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Cloning, sequencing and biochemical characterization of RecA and RuvB from divergent thermophilic bacteria.
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Identifier
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AAI9808017
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identifier
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9808017
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Creator
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Tong, Jie.
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Contributor
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Adviser: James G. Wetmur
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology
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Abstract
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The recA gene of Thermus thermophilus was cloned, sequenced and expressed in Escherichia coli. Three other thermostable RecA proteins were cloned by other members of our laboratory group from the highly-divergent thermophilic eubacteria Thermus aquaticus, Thermotoga maritima and Aquifex pyrophilus. In contrast to the E. coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70{dollar}\sp\circ{dollar}C. In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit.;The ruvB genes of the highly divergent thermophilic eubacteria T. thermophilus and T. maritima were cloned, sequenced and expressed in Escherichia coli. Both thermostable RuvB proteins were purified to homogeneity. Like E. coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima ({dollar}\ge{dollar}70{dollar}\sp\circ{dollar}C). In the absence of ATP, T. thermophilus RuvS protein bound to linear double-stranded DNA with a preference for the ends. Addition of ATP or ATP{dollar}\gamma{dollar}S destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA. Expression of thermostable T. thermophilus RuvB protein in the E. coli ruvBrecG mutant strain N3395 partially complemented the UV sensitive phenotype suggesting that T. thermophilus RuvB protein has a function similar to E. coli RuvB in vivo.;To solve the crystal structure, T. maritima and T. thermophilus RuvB proteins were expressed and purified from the E. coli strain BL21(DE3) pLysS. Crystallization of the T. maritima and T. thermophilus RuvB proteins was achieved at Scripps Research Institute by both the hanging-drop and sitting-drop vapor diffusion techniques. T. maritima and T. thermophilus RuvB crystals gave diffraction data to at least 2.2 A and 3.6 A, respectively. Two T. maritima RuvB cysteine mutants, A308C and N32C, were generated for derivatization by mercurials. Both mutant proteins were expressed, purified and crystallized. Selenomethionine-substituted RuvB protein was generated for multiwavelength anomalous diffraction phasing. Based on mass spectra data, the level of selenomethionine substitution was almost complete.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
