DNA Polymerases

Eun, Hyone‐Myong

doi:10.1016/b978-012243740-3/50009-0

Cited by 22 publications

(27 citation statements)

References 470 publications

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“…It should be mentioned that RNase A cleaved mostly after U and C residues that were followed by As, as judged by the results of RNase A digestion under denaturing conditions (C/U ladder, Figure 7A, lane 8). Indeed, it has been determined that the residue located 3′ to C and U affects the k cat of RNase A by a factor of >100 between CpA and CpU (80). Therefore, we could not determine structure status of pyrimidines followed by a residue other than A. Interestingly, RNase A cleaved after 60U under native but not denaturing conditions, indicating that this residue is highly exposed to the enzyme in the folded RNA (Figure 7A, lane 7, marked by a square).…”

Section: Resultsmentioning

confidence: 99%

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Singh

Androphy

2006

Nucleic Acids Research

194

215

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Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.

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Section: Resultsmentioning

confidence: 99%

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Singh

Androphy

2006

Nucleic Acids Research

194

215

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“…It cannot continuously incorporate nucleotides without dissociating from the primer template. Thioredoxin significantly increases its affinity for the template, so that the DNA synthesis becomes processive for thousands of nucleotides (15, 16). By contrast, the Klenow fragment incorporates about 15 bases before dissociating, T4 DNA polymerase - about 10 bases, T5 DNA polymerase - about 180 bases (17, 18).…”

Section: Introductionmentioning

confidence: 99%

“…By contrast, the Klenow fragment incorporates about 15 bases before dissociating, T4 DNA polymerase - about 10 bases, T5 DNA polymerase - about 180 bases (17, 18). In fact T7 DNA polymerase is one of the most processive thermolabile DNA polymerases, with higher processivity then Pol I, Klenow fragment or T4 DNA polymerase (15, 16). Importantly, T7 DNA polymerase efficiently incorporates not only normal dNTPs, but also biotinylated nucleotides (3).…”

Section: Introductionmentioning

confidence: 99%

“…This is not likely to happen in fixed DNA in tissue sections, because it occurs when ssDNA loops back upon itself and hybridizes to the same strand. Any unpaired nucleotides at the 3’ terminus are then removed by the 3’→5’ exonuclease activity until a base-paired terminus is reached and is used to prime the new strand synthesis (15, 19). …”

Section: Introductionmentioning

confidence: 99%

“…In the absence of nucleoside triphosphates, the enzyme initiates DNA hydrolysis at nicks and 3’OH termini in DNA (15). Starting from the available 3’OH ends, it will reduce 3’ overhangs and blunt-ended DNA breaks into long 5’overhangs.…”

Section: Introductionmentioning

confidence: 99%

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In Situ Labeling of DNA Breaks and Apoptosis by T7 DNA Polymerase

Didenko

2010

Methods in Molecular Biology

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The native T7 DNA polymerase is a fast and highly processive enzyme which can be used for in situ detection of apoptosis and various types of DNA breaks. The technique is quick and simple, and was shown to label earlier stages of apoptosis compared to the terminal transferase technique. The in situ labeling applications of T7 DNA polymerase are presented and summarized from the DNA damage detection standpoint. The detailed protocols are provided together with the discussion of their advantages and limitations.

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Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel Streptomyces sp. strain from a Saharan soil

Djinni

Defant

Djoudi

et al. 2019

Heliyon

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The novel bioactive actinobacterial strain GSBNT10 obtained from a Saharan soil, was taxonomically characterized using a polyphasic approach. 16S rRNA gene sequence analysis supported the classification of the isolate within the genus Streptomyces indicating it as a novel species. The major metabolite responsible of the bioactivity was purified and structurally characterized as actinomycin D (act-D) by mass spectrometric and nuclear magnetic resonance analyses Plackett-Burman design (PBD) and response surface methodology (RSM) were applied in order to optimize the medium formulation for the production of this bioactive metabolite. By PBD experiments, NaNO 3 , K 2 HPO 4 and initial pH value were selected as significant variables affecting the metabolite production. Central Composite Design (CCD) showed that adjustment of the fermentative medium at pH 8.25, K 2 HPO 4 at 0.2 gL -1 and NaNO 3 at 3.76 gL -1 were the values suiting the production of act-D. Moreover, the results obtained by the statistical approach were confirmed by act-D detection using the HPLC equipped with a diode array detector and coupled online with electrospray-mass spectrometry (ESIMS) technique. act-D production was highly stimulated, obtaining a good yield (656.46 mgL -1 ) which corresponds to a 58.56% increase compared with the non-optimized conditions and data from LC-ESIMS technique efficiently confirmed the forecast from RSM.

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DNA Polymerases

Cited by 22 publications

References 470 publications

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

In Situ Labeling of DNA Breaks and Apoptosis by T7 DNA Polymerase

Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel Streptomyces sp. strain from a Saharan soil

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