Assay of Restriction Endonucleases Using Oligonucleotides

Connolly, Bernard A.; Liu, Hsiao-Hui; Parry, D. C.; Engler, Lisa E.; Kurpiewski, Michael R.; Jen‐Jacobson, Linda

doi:10.1385/1-59259-208-2:465

Cited by 6 publications

(5 citation statements)

References 11 publications

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“…We therefore utilized a second, independent method for the determination of K d values for the M.EcoRII/AdoHcy/DNA complexes using a competition titration-fluorescence polarization technique. This is a true equilibrium method since the experiments are carried out in aqueous solutions and are not influenced by potential gel cage effects, irreversible protein-DNA dissociation, or effects associated with varying enzyme concentrations within the gel (41). The fluorescence of the fluorescein-tagged duplex FAM-U was easily measurable, and the reproducibility was excellent (within a few percent).…”

Section: Resultsmentioning

confidence: 99%

“…The 18-bp FAM-U duplex showed only a small decrease in the fluorescence yield upon binding to M.EcoRII. This observation suggests that the effect of the MTase on the fluorescence of the fluorescein fluorophore is small, and its presence at the 5′-terminus does not significantly interfere with the binding of the 18-mer substrates (41). This small change in the fluorescence yields was therefore neglected in the calculations of the K 1 and K 2 values.…”

Section: Resultsmentioning

confidence: 99%

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Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease

et al. 2004

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DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease

et al. 2004

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“…After 1 h incubation at 37°C, the samples were processed as described above. The data were fitted using the one-site competitive binding model (35).…”

Section: Methodsmentioning

confidence: 99%

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Kuzmenko

Yudin

Ryazansky

et al. 2019

Nucleic Acids Research

169

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Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro . However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5′-nucleotide in guide DNA and can use not only 5′-phosphorylated but also 5′-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3′-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo .

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“…50,53,54 All equilibrium binding reactions were carried out in binding buffer (10 mM bisTris-propane, 100 mM dithiothreitol, 1 mM EDTA, 100 mg/ml bovine serum albumin, pH 7.3) plus 0.22 M KCl.…”

Section: Equilibrium Binding and Cleavage Kineticsmentioning

confidence: 99%

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

Sapienza

Torre

McCoy

et al. 2005

Journal of Molecular Biology

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Assay of Restriction Endonucleases Using Oligonucleotides

Cited by 6 publications

References 11 publications

Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease

Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

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