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Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI.

Yang Z, Horton JR, Maunus R, Wilson GG, Roberts RJ, Cheng X - Nucleic Acids Res. (2005)

Bottom Line: Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C/CGG) and binds to that sequence crystallographically as a monomer.Examining the protein-protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer.A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine 1510 Clifton Road, Atlanta, GA 30322, USA.

ABSTRACT
HinP1I, a type II restriction endonuclease, recognizes and cleaves a palindromic tetranucleotide sequence (G/CGC) in double-stranded DNA, producing 2 nt 5' overhanging ends. Here, we report the structure of HinP1I crystallized as one protein monomer in the crystallographic asymmetric unit. HinP1I displays an elongated shape, with a conserved catalytic core domain containing an active-site motif of SDX18QXK and a putative DNA-binding domain. Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C/CGG) and binds to that sequence crystallographically as a monomer. Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements. Examining the protein-protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer. A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

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HinP1I solution properties (A) Overlay of chromatographs from elution experiments on a Superdex 75 HR 10/30 sizing column. Blue: elution of three molecular weight standard proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa) and BSA (66.0 kDa). Red: elution of HinPI1 after loading 100 μl at ∼3.7 mg/ml concentration. Magenta: elution of HinPI1 after loading 100 μl at ∼75 mg/ml concentration. (B) HinPI1 in the presence of increasing amounts of glutaraldehyde. HinPI1 (∼1.9 mg/ml) was treated with glutaraldehyde and incubated at room temperature for 1 h (total volume 20 μl). The reaction was stopped by the addition of 10 μl of 1 M glycine. An aliquot of 15 μl of 3× loading buffer was added to these samples and subsequently 20 μl of this final solution was loaded onto a 13% SDS–PAGE gel (Coomassie stain).
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fig6: HinP1I solution properties (A) Overlay of chromatographs from elution experiments on a Superdex 75 HR 10/30 sizing column. Blue: elution of three molecular weight standard proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa) and BSA (66.0 kDa). Red: elution of HinPI1 after loading 100 μl at ∼3.7 mg/ml concentration. Magenta: elution of HinPI1 after loading 100 μl at ∼75 mg/ml concentration. (B) HinPI1 in the presence of increasing amounts of glutaraldehyde. HinPI1 (∼1.9 mg/ml) was treated with glutaraldehyde and incubated at room temperature for 1 h (total volume 20 μl). The reaction was stopped by the addition of 10 μl of 1 M glycine. An aliquot of 15 μl of 3× loading buffer was added to these samples and subsequently 20 μl of this final solution was loaded onto a 13% SDS–PAGE gel (Coomassie stain).

Mentions: While a monomer/dimer equilibrium was observed for MspI in analytical ultracentrifuge experiments (5), we used several alternative ways to establish whether HinP1I exists as a monomer, dimer or tetramer in solution. First, gel filtration suggests that HinP1I has an apparent molecular weight of a monomer. On a Superdex75 column (Pharmacia), Hinp1I was loaded onto the column at an initial concentration of 3.7 mg/ml and eluted at an elution volume of 10.48, equivalent to a 28 kDa globular protein (Figure 6A). This is similar to the calculated molecular weight of 28.75 kDa. Only at a loading concentration of ∼75 mg/ml was a small fraction of HinP1I eluted at a size close to that of a dimer at ∼59 kDa (Figure 6A).


Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI.

Yang Z, Horton JR, Maunus R, Wilson GG, Roberts RJ, Cheng X - Nucleic Acids Res. (2005)

HinP1I solution properties (A) Overlay of chromatographs from elution experiments on a Superdex 75 HR 10/30 sizing column. Blue: elution of three molecular weight standard proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa) and BSA (66.0 kDa). Red: elution of HinPI1 after loading 100 μl at ∼3.7 mg/ml concentration. Magenta: elution of HinPI1 after loading 100 μl at ∼75 mg/ml concentration. (B) HinPI1 in the presence of increasing amounts of glutaraldehyde. HinPI1 (∼1.9 mg/ml) was treated with glutaraldehyde and incubated at room temperature for 1 h (total volume 20 μl). The reaction was stopped by the addition of 10 μl of 1 M glycine. An aliquot of 15 μl of 3× loading buffer was added to these samples and subsequently 20 μl of this final solution was loaded onto a 13% SDS–PAGE gel (Coomassie stain).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074309&req=5

fig6: HinP1I solution properties (A) Overlay of chromatographs from elution experiments on a Superdex 75 HR 10/30 sizing column. Blue: elution of three molecular weight standard proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa) and BSA (66.0 kDa). Red: elution of HinPI1 after loading 100 μl at ∼3.7 mg/ml concentration. Magenta: elution of HinPI1 after loading 100 μl at ∼75 mg/ml concentration. (B) HinPI1 in the presence of increasing amounts of glutaraldehyde. HinPI1 (∼1.9 mg/ml) was treated with glutaraldehyde and incubated at room temperature for 1 h (total volume 20 μl). The reaction was stopped by the addition of 10 μl of 1 M glycine. An aliquot of 15 μl of 3× loading buffer was added to these samples and subsequently 20 μl of this final solution was loaded onto a 13% SDS–PAGE gel (Coomassie stain).
Mentions: While a monomer/dimer equilibrium was observed for MspI in analytical ultracentrifuge experiments (5), we used several alternative ways to establish whether HinP1I exists as a monomer, dimer or tetramer in solution. First, gel filtration suggests that HinP1I has an apparent molecular weight of a monomer. On a Superdex75 column (Pharmacia), Hinp1I was loaded onto the column at an initial concentration of 3.7 mg/ml and eluted at an elution volume of 10.48, equivalent to a 28 kDa globular protein (Figure 6A). This is similar to the calculated molecular weight of 28.75 kDa. Only at a loading concentration of ∼75 mg/ml was a small fraction of HinP1I eluted at a size close to that of a dimer at ∼59 kDa (Figure 6A).

Bottom Line: Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C/CGG) and binds to that sequence crystallographically as a monomer.Examining the protein-protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer.A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine 1510 Clifton Road, Atlanta, GA 30322, USA.

ABSTRACT
HinP1I, a type II restriction endonuclease, recognizes and cleaves a palindromic tetranucleotide sequence (G/CGC) in double-stranded DNA, producing 2 nt 5' overhanging ends. Here, we report the structure of HinP1I crystallized as one protein monomer in the crystallographic asymmetric unit. HinP1I displays an elongated shape, with a conserved catalytic core domain containing an active-site motif of SDX18QXK and a putative DNA-binding domain. Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C/CGG) and binds to that sequence crystallographically as a monomer. Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements. Examining the protein-protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer. A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

Show MeSH