[6] Efficient site-directed mutagenesis using uracil-containing DNA

Kunkel, Thomas A.; Bębenek, Katarzyna; McClary, John

doi:10.1016/0076-6879(91)04008-c

Cited by 668 publications

(449 citation statements)

References 12 publications

Supporting

Mentioning

448

Contrasting

Order By: Relevance

“…Hexahistidine-tagged CED-9 (amino acids 68-251) was produced using the pET 15b vector (Novagen). Mutagenesis of both CED-4 and CED-9 was performed using the method of Kunkel et al 33 All constructs were confirmed by bidirectional automated DNA sequencing.…”

Section: Plasmid Constructionmentioning

confidence: 99%

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

et al. 2005

View full text Add to dashboard Cite

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-offunction CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

show abstract

Section: Plasmid Constructionmentioning

confidence: 99%

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

et al. 2005

View full text Add to dashboard Cite

show abstract

“…Site-directed Mutagenesis-Site-directed mutants were obtained by oligonucleotide mutagenesis using the method of Kunkel et al (38). To generate the single-stranded DNA needed for mutagenesis, a BglII fragment of the PvuII gene (Asp 5 -Leu 120 ) was subcloned into LITMUS 28 (New England Biolabs Inc.).…”

Section: Methodsmentioning

confidence: 99%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...

show abstract

“…The V32D/F57H double mutant was created by single-stranded mutagenesis (11). Plasmids were isolated and screened via restriction digest with the enzyme DraI.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Ory

Kane

Simpson

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

A number of crystallographic studies of the adipocyte lipid-binding protein have established that the fatty acid-binding site is within an internalized water-filled cavity. The same studies have also suggested the existence of a region physically distinct from the fatty acid-binding site which connects the cavity of the protein with the external solvent, hereafter referred to as the portal. In an effort to examine the portal region, we have used site-directed mutagenesis to introduce the mutations V32D/F57H into the murine ALBP cDNA. Mutant protein has been isolated, crystallized, and its stability and binding properties studied by biochemical methods. As assessed by guanidine-HCl denaturation, the mutant form exhibited a slight overall destabilization relative to the wild-type protein under both acid and alkaline conditions. Accessibility to the cavity in both the mutant and wild-type proteins was observed by stopped-flow analysis of the modification of a cavity residue, Cys 117 , by the sulfhydryl reactive agent 5,5 -dithiobis(2-nitrobenzoic acid) at pH 8.5. Cys 117 of V32D/F57H ALBP was modified 7-fold faster than the wild-type protein. The ligand binding properties of both the V32D/F57H mutant and wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation constants for 1-anilinonaphthalene-8-sulfonic acid were approximately 9 -10-fold greater than the wild-type protein, independent of pH. In addition, there is a 6-fold increase in the K d for oleic acid for the portal mutant relative to the wild-type at pH 8.0. To study the effect of pH on the double mutant, it was crystallized and analyzed in two distinct space groups at pH 4.5 and 6.4. While in general the differences in the overall main chain conformations are negligible, changes were observed in the crystallographic structures near the site of the mutations. At both pH values, the mutant side chains are positioned somewhat differently than in wild-type protein. To ensure that the mutations had not altered ionic conditions near the binding site, the crystallographic coordinates were used to monitor the electrostatic potentials from the head group site to the positions near the portal region. The differences in the electrostatic potentials were small in all regions, and did not explain the differences in ligand affinity. We present these results within the context of fatty acid binding and suggest lipid association is more complex than that described within a single equilibrium event.

show abstract

[6] Efficient site-directed mutagenesis using uracil-containing DNA

Cited by 668 publications

References 12 publications

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Contact Info

Product

Resources

About