Ca<sup>2+</sup> Binding in the Active Site of HincII:  Implications for the Catalytic Mechanism<sup>,</sup>

Etzkorn, C.; Horton, Nancy C.

doi:10.1021/bi0490082

Cited by 28 publications

(46 citation statements)

References 62 publications

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“…It is noteworthy that the single-turnover rate constant of IN is particularly slow, for example about one thousandth that for restriction enzyme HincII (53). This indicates that a pre-catalysis step has a limiting effect independent from that for product release.…”

Section: Discussionmentioning

confidence: 99%

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Guiot

Carayon

Delelis

et al. 2006

Journal of Biological Chemistry

137

143

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The 3-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in realtime. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN⅐DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3-processing, with a dimer of dimers responsible for the subsequent full integration.The integration of a DNA copy of the HIV-1 2 genome into the host genome is a crucial step in the life cycle of the retrovirus. Integrase (IN) is responsible for the two consecutive reactions that constitute the overall integration process. The first of these two reactions is 3Ј-processing, which involves cleavage of the 3Ј-terminal GT dinucleotide at each extremity of the viral DNA. The hydroxyl groups of newly recessed 3Ј-ends are then used in the second reaction, strand transfer, for the covalent joining of viral and target DNAs, resulting in full-site integration. IN is sufficient for catalysis of the 3Ј-processing reaction in vitro, using short-length oligodeoxynucleotides (ODNs) that mimic one viral long terminal repeat (LTR) in the presence of the metallic cofactor Mg 2ϩ . This reaction generates two products: the viral DNA containing the recessed extremity and the GT dinucleotide. One of the two products, the processed viral DNA, as well as the target DNA serve as substrates for the subsequent joining reaction.IN belongs to the superfamily of polynucleotidyl transferases. Its catalytic core domain contains a triad of acidic residues constituting the D,D-35-E motif, which is strictly required for catalysis. The catalytic core establishes specific contacts with the viral DNA and, together with the C-terminal domain, is involved in DNA binding (1-4). ...

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Section: Discussionmentioning

confidence: 99%

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Guiot

Carayon

Delelis

et al. 2006

Journal of Biological Chemistry

137

143

View full text Add to dashboard Cite

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“…The putative nucleophilic water molecule was also observed in the structure of HincII and was within hydrogen-bonding distance of the conserved active site lysine (Lys-129) as well as the oxygen of the 3Ј-phosphate group of scissile phosphate (6). The importance of 3Ј-phosphate during the activation of water molecule was discussed (6). NW in the EcoO109I also interacts with the oxygen of 3Ј-phosphate group of scissile phosphate.…”

Section: Resultsmentioning

confidence: 93%

“…The general base activation is also aided by polarization of the water molecule through its coordination to the divalent metal ion. The putative nucleophilic water molecule was also observed in the structure of HincII and was within hydrogen-bonding distance of the conserved active site lysine (Lys-129) as well as the oxygen of the 3Ј-phosphate group of scissile phosphate (6). The importance of 3Ј-phosphate during the activation of water molecule was discussed (6).…”

Section: Resultsmentioning

confidence: 94%

“…The Ca 2ϩ in the structure of HincII is also coordinated square-bipyramidally with six oxygen atoms including the scissile phosphate similar to the structure of EcoO109I. The direct ligation by the metal ion to the scissile phosphate might be ubiquitous interaction in the metal ion-assisted reaction by type II restriction endonuclease, which produces 5Ј-or 3Ј-overhang DNA (6).…”

Section: Resultsmentioning

confidence: 96%

“…Most recently, the crystal structure of HincII bound to Ca 2ϩ and cognate DNA. The DNA is uncleaved, and one calcium ion is bound per active site (6). The Ca 2ϩ in the structure of HincII is also coordinated square-bipyramidally with six oxygen atoms including the scissile phosphate similar to the structure of EcoO109I.…”

Section: Resultsmentioning

confidence: 99%

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Crystal Structures of Type II Restriction Endonuclease EcoO109I and Its Complex with Cognate DNA

Hashimoto

Shimizu

Imasaki

et al. 2005

Journal of Biological Chemistry

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EcoO109I is a type II restriction endonuclease that recognizes the DNA sequence of RGGNCCY. Here we describe the crystal structures of EcoO109I and its complex with DNA. A comparison of the two structures shows that the catalytic domain moves drastically to capture the DNA. One metal ion and two water molecules are observed near the active site of the DNA complex. The metal ion is a Lewis acid that stabilizes the pentavalent phosphorus atom in the transition state. One water molecule, activated by Lys-126, attacks the phosphorus atom in an S N 2 mechanism, whereas the other water interacts with the 3-leaving oxygen to donate a proton to the oxygen. EcoO109I is similar to EcoRI family enzymes in terms of its DNA cleavage pattern and folding topology of the common motif in the catalytic domain, but it differs in the manner of DNA recognition. Our findings propose a novel classification of the type II restriction endonucleases and lead to the suggestion that EcoO109I represents a new subclass of the EcoRI family.Bacteria have evolved a mechanism to protect themselves from viral infection. Restriction endonucleases (REases) 1 provide an anti-viral protection for bacteria by degrading the foreign DNA of invading bacteriophages. The enzymes recognize specific nucleotide sequences and cleave both strands of DNA. To date, more than 3,500 REases have been characterized and classified into four types, I, II, III, and IV (1). Of these types, type II REases are widely used in genetic technology and are the most well studied.Type II REases generally recognize 4 -8 base pairs of doublestranded DNA and hydrolyze phosphodiester bonds within the recognition sequences. The amino acid sequences are not homologous with the exception of the active site sequence. The active sites of the type II enzymes have a signature sequence PD(X) n DXK, in which four residues (Pro, Asp, Asp, and Lys; n ϭ 1 (PvuII) to ϳ49 (Bse634I) (2)) are weakly conserved and two separated acidic residues are usually followed by a basic residue. The active site structures thus share a common structural motif consisting of a five-stranded ␤-sheet flanked with ␣-helices (3-5). Specific divalent metal cations such as Mg 2ϩ are required to express the enzymatic activities and are coordinated to the conserved acidic residues during the catalytic reaction. However, details of the mechanism of the catalytic reaction are not fully understood (2).Type II REases have been classified into two families, EcoRI and EcoRV, based on their DNA cleavage pattern. The EcoRI family produces 5Ј-overhang DNA, whereas the EcoRV family produces blunt-end DNA. From a structural viewpoint, the EcoRI family approaches DNA from the major groove side, whereas the EcoRV family approaches from the minor groove side. Moreover, the folding topology of the five-stranded ␤-sheet in the common structural motif in the active site differs between the two families (7) where the topology of four ␤-strands are absolutely conserved in the common motif but the fifth ␤-strand is oppositely oriented (8).To ...

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A “moving metal mechanism” for substrate cleavage by the DNA repair endonuclease APE‐1

et al. 2007

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Apurinic/apyrimidinic endonuclease (APE-1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild-type structure of the metal-free APE1/damaged-DNA complex (1DE8). A grid search with one Mg2+ ion located two low energy clusters of Mg2+ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg2+ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a "moving metal mechanism" in which one Mg2+ ion moves from the B- (more buried) to the A-site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in-line cleavage reaction. Experimental results, which show competition between Ca2+ and Mg2+ for catalyzing the reaction, and high concentrations of Mg2+ are inhibitory, indicate that both sites cannot be simultaneously occupied for maximal activity.

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Ca²⁺ Binding in the Active Site of HincII: Implications for the Catalytic Mechanism^,

Cited by 28 publications

References 62 publications

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Crystal Structures of Type II Restriction Endonuclease EcoO109I and Its Complex with Cognate DNA

A “moving metal mechanism” for substrate cleavage by the DNA repair endonuclease APE‐1

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Ca2+ Binding in the Active Site of HincII: Implications for the Catalytic Mechanism,

Cited by 28 publications

References 62 publications

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Crystal Structures of Type II Restriction Endonuclease EcoO109I and Its Complex with Cognate DNA

A “moving metal mechanism” for substrate cleavage by the DNA repair endonuclease APE‐1

Contact Info

Product

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About

Ca²⁺ Binding in the Active Site of HincII: Implications for the Catalytic Mechanism^,