Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Moreira, Leonilde M.; Costa, Milton S. da; Sá‐Correia, Isabel

doi:10.1007/bf02568728

Cited by 6 publications

(1 citation statement)

References 22 publications

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“…Although the concentration of each deoxynucleotide including dTTP is 25-100 M in organisms, that of each ribonucleotides, including rUTP, is approximately 1000 M (19). We estimate that if WT Taq pol I were to replicate the entire 2 ϫ 10 6 base pair eubacteria genome (20), assuming the rUTP concentration is 10-fold greater than that of dTTP, WT Taq pol I should introduce approximately 10 rUMP into genomic DNA per replication cycle, and these substitutions would presumably be corrected by DNA repair. Other polymerases from prokaryotic pol I, eukaryotic pol ␣, and RT families also discriminate against ribouracil (Table I).…”

Section: Discussionmentioning

confidence: 99%

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

Patel

Loeb

2000

Journal of Biological Chemistry

101

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DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10 3 -fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.Both DNA and RNA polymerase can catalyze chain elongation reaction guided by single-stranded DNA templates to generate polynucleotide products (1). The order of nucleotide addition proceeds in a 5Ј 3 3Ј direction via metal-mediated phosphoryl transfer reaction resulting in the formation of phosphodiester bond and release of pyrophosphate (2). In addition, both DNA and RNA polymerases resemble in morphology a cupped human right hand and bind DNA template and the incoming nucleotide within the active site cleft (3, 4). DNA polymerases differ from RNA polymerases in utilizing 2Ј-deoxynucleotides (dTTP, dCTP, dGTP, and dATP) rather than ribonucleotides (rUTP, rCTP, rGTP, and rATP). A detailed analysis of the polymerase active site is crucial to understanding how these polymerases distinguish between dNTPs and rNTPs, as well as to provide insights on how the two types of polymerases co-evolved to adopt similar mechanisms.Despite the similarity in protein structure and function, there is almost no sequence identity between DNA and RNA polymerases. For example, nearly all of the over 40 prokaryotic and eubacteria DNA pol Is 1 sequenced (including Thermus aquaticus pol I, Chlamydia trachomatis pol I, and Escherichia coli pol I) contain the DYSQIELR sequence within the dNTP binding site (motif A; Ref. 5), yet RNA pols only have in common the catalytically essential aspartic acid residue (6). If evolution proceeded from an "RNA world" containing RNA synthesizing enzymes to a "DNA world" with genomes replicated by DNA synthesizing enzymes (7, 8), it might be possible to gain insights into this process by substituting random sequences within the active site ...

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Section: Discussionmentioning

confidence: 99%

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

Patel

Loeb

2000

Journal of Biological Chemistry

101

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Thermus

Costa

Nobre

Rainey

2015

Bergey's Manual of Systematics of Archaea and Bacteria

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Ther' mus . Gr. adj. thermos hot; M.L. masc. n. Thermus to indicate an organism living in hot places. “Deinococcus‐Thermus” / Deinococci / Thermales / Thermaceae / Thermus Straight rods , 0.5–0.8 µm in diameter; the cell length is variable. Short filaments are also formed under some culture conditions. Some strains have a stable filamentous morphology. Nonmotile ; do not possess flagella. Endospores are not observed. Stain Gram‐negative. Most strains form yellow‐pigmented colonies , some strains are nonpigmented. Aerobic with a strictly respiratory type of metabolism, but some strains grow anaerobically with nitrate and nitrite as terminal electron acceptors. Oxidase positive and catalase positive. Thermophilic , with an optimum growth temperature of about 70–75°C; most strains have a maximum growth temperature below 80°C, but some strains grow at higher temperatures. The optimum pH is about 7.8. Menaquinone 8 is the predominant respiratory quinone; ornithine is the principal diamino acid of the peptidoglycan. One major phospholipid and one major glycolipid dominate the polar lipid pattern on thin‐layer chromatography. Additional phospholipids and glycolipids are minor components. Fatty acids are predominantly iso‐ and anteiso‐branched ; branched chain 3‐hydroxy fatty acids are present in some strains. Proteins and peptides are hydrolyzed by all strains. Starch is hydrolyzed by some strains. Monosaccharides, disaccharides, amino acids, and organic acids are used as sole carbon and energy sources. The utilization of pentoses and polyols is very rare. Most strains require yeast extract or cofactors for growth. Found in hydrothermal areas with neutral to alkaline pH, also commonly isolated from man‐made thermal environments. The mol % G + C of the DNA is : 57–65. Type species : Thermus aquaticus Brock and Freeze 1969, 295.

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Thermus

Albuquerque¹,

Rainey²,

Costa³

2018

Bergey's Manual of Systematics of Archaea and Bacteria

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Ther'mus. Gr. adj. thermos hot; N.L. masc. n. Thermus to indicate an organism living in hot places. “Deinococcus‐Thermus” / Deinococci / Thermales / Thermaceae / Thermus Straight rods; the cell length is variable, 0.2–1.7 µm in width. Filaments are also formed; some strains have a stable filamentous morphology. Nonmotile; do not possess flagella. Endospores are not observed. Stain Gram‐negative. Most strains form yellow‐pigmented colonies; some strains are nonpigmented. Thermophilic, with an optimum growth temperature of about 65–75°C; most strains have a maximum growth temperature below 80°C, but some strains grow at slightly higher temperatures. The optimum pH for growth is about 7.0–8.5. Growth is chemoorganotrophic; some strains are mixotrophic using arsenic, sulfur or thiosulfate as electron donors. Aerobic with a strictly respiratory type of metabolism; some strains grow anaerobically with nitrate, nitrite, and iron as terminal electron acceptors; some strains can also reduce gold, chromate, and uranium under aerobic and anaerobic conditions. Oxidase positive, most strains are catalase positive. Strains generally require yeast extract or cofactors for growth. Sugars, amino acids, organic acids, and polyols are used as sole organic carbon and energy sources. Ornithine is the principal diamino acid of the peptidoglycan. Menaquinone 8 (MK‐8) is the predominant respiratory quinone. One major phospholipid and one major glycolipid dominate the polar lipid pattern on thin‐layer chromatography. Additional phospholipids and glycolipids are minor components. Phospholipids and glycolipids are primarily glycerol based, but some strains also possess 1,2‐long‐chain diol‐based lipids. Fatty acids are predominantly iso‐ and anteiso‐ branched; branched‐chain 3‐hydroxy fatty acids are present in some strains. Found in hydrothermal areas with neutral to alkaline pH, also commonly isolated from man‐made thermal environments. DNA G + C content (mol%) : 63–71 (Tm or HPLC). Type species : Thermus aquaticus Brock and Freeze 1969, 295 AL .

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Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Cited by 6 publications

References 22 publications

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

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