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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.Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements.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.Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements.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