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1.
Nucleic Acids Res ; 47(1): 450-467, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30395313

ABSTRACT

BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).


Subject(s)
DNA Cleavage , DNA Restriction Enzymes/chemistry , Holoenzymes/chemistry , Protein Subunits/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , DNA Restriction Enzymes/genetics , DNA Restriction Enzymes/isolation & purification , Escherichia coli/enzymology , Holoenzymes/genetics , Holoenzymes/isolation & purification , Mutation , Peptides/chemistry , Protein Multimerization , Protein Subunits/genetics , Protein Subunits/isolation & purification
2.
Nucleic Acids Res ; 45(3): 1516-1528, 2017 02 17.
Article in English | MEDLINE | ID: mdl-28180307

ABSTRACT

R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar 'PD-D/ExK' restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.


Subject(s)
DNA/chemistry , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , AT Rich Sequence , Amino Acid Sequence , Base Pairing , Binding Sites , Crystallography, X-Ray , DNA/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Models, Molecular , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , Structural Homology, Protein , Substrate Specificity
3.
Genome Announc ; 3(1)2015 Feb 19.
Article in English | MEDLINE | ID: mdl-25700417

ABSTRACT

Bacillus strain X1 is the source strain for the restriction enzyme BstXI. Its complete sequence and full methylome was determined using single-molecule real-time (SMRT) sequencing.

4.
Protein Expr Purif ; 58(1): 42-52, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18164625

ABSTRACT

BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).


Subject(s)
Deoxyribonucleases, Type II Site-Specific , Amino Acid Sequence , Bacillus megaterium/enzymology , Cloning, Molecular , DNA Cleavage , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli/genetics , Gene Expression , Molecular Sequence Data , Plasmids , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , SOS Response, Genetics , Temperature
5.
J Mol Biol ; 348(3): 631-40, 2005 May 06.
Article in English | MEDLINE | ID: mdl-15826660

ABSTRACT

The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R(1) subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC--TGAGG, while those in which the catalytic site in the R(2) subunit remained functional nicked the top strand, producing CC--TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification.


Subject(s)
Bacterial Proteins/metabolism , Base Sequence , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Catalytic Domain , Cations/metabolism , DNA/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Subunits/genetics , Protein Subunits/metabolism , Sequence Alignment , Sequence Analysis, DNA , Substrate Specificity
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