How Well Should the Active Site and the Specific Recognition Be Defined for Proficient Catalysis? – Effective and Cooperative Polyphenol/Catechol Oxidation and Oxidative DNA Cleavage by a Copper(II)-Binding and H-Bonding Copolymer

2008 ◽  
Vol 2008 (16) ◽  
pp. 2584-2592 ◽  
Author(s):  
Vasiliki Lykourinou ◽  
Ahmed I. Hanafy ◽  
Giordano F. Z. da Silva ◽  
Kirpal S. Bisht ◽  
Randy W. Larsen ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Specific Recognition ◽  
Catechol Oxidation ◽  
Oxidative Dna Cleavage

scholarly journals Structure-Function Analysis of IntDOT

2009 ◽  
Vol 192 (2) ◽  
pp. 575-586 ◽  
Author(s):  
Seyeun Kim ◽  
Brian M. Swalla ◽  
Jeffrey F. Gardner
Keyword(s):  
Homology Modeling ◽  
Protein Interactions ◽  
Active Site ◽  
Dna Cleavage ◽  
Function Analysis ◽  
Indicator Strain ◽  
Amino Terminal ◽  
Dna Ligation ◽  
Polypeptide Backbone

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

Oxidative DNA cleavage by copper ternary complexes of 1,10-phenanthroline and ethylenediamine-sulfonamide derivatives

Polyhedron ◽  
2009 ◽  
Vol 28 (13) ◽  
pp. 2537-2544 ◽  
Author(s):  
Andreea Bodoki ◽  
Adriana Hangan ◽  
Luminita Oprean ◽  
Gloria Alzuet ◽  
Alfonso Castiñeiras ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Ternary Complexes ◽  
Oxidative Dna Cleavage

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

Roles of Active-Site Amino Acid Residues in Specific Recognition of DNA Lesions by Human 8-Oxoguanine-DNA Glycosylase (OGG1)

2019 ◽  
Vol 123 (23) ◽  
pp. 4878-4887 ◽  
Author(s):  
Timofey E. Tyugashev ◽  
Yury N. Vorobjev ◽  
Alexandra A. Kuznetsova ◽  
Maria V. Lukina ◽  
Nikita A. Kuznetsov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Amino Acid Residues ◽  
Specific Recognition

Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions

2011 ◽  
Vol 6 (9) ◽  
pp. 934-942 ◽  
Author(s):  
Kommireddy Vasu ◽  
Matheshwaran Saravanan ◽  
Valakunja Nagaraja
Keyword(s):  
Transition Metal ◽  
Metal Ions ◽  
Active Site ◽  
Dna Cleavage ◽  
Alkaline Earth ◽  
Transition Metal Ions

scholarly journals Fluorophore ATCUN complexes: combining agent and probe for oxidative DNA cleavage

2015 ◽  
Vol 51 (62) ◽  
pp. 12395-12398 ◽  
Author(s):  
C. Wende ◽  
N. Kulak
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Fluorescent Reporter ◽  
Dna Cleaving ◽  
Oxidative Dna Cleavage

A Cu(ii)-based peptidic DNA cleaving agent equipped with a Cu(ii)-sensing fluorescent reporter allows monitoring the fate of the nucleolytic metal ion.

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