Summary: Restriction endonuclease BglII
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BglII Edit Wikipedia article
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|Restriction endonuclease BglII|
structure of restriction endonuclease BstYI bound to non-cognate DNA
|SCOPe||1dfm / SUPFAM|
Like most type II restriction enzymes, BglII consists of two identical subunits that form a homodimer around the DNA double helix. Each monomer is 223 amino acids and symmetrically bind both sides of the unique palindromic nucleotide sequence AGATCT, cleaving the scissile phosphodiester bond between the first Adenine and Guanine nucleotides on both strands of the DNA molecule, creating sticky ends with 5' end overhangs.
Being a type II restriction enzyme, BglII does not require ATP (adenosine triphosphate) for its enzymatic function, but only requires association with a divalent metal cation, most likely Mg2+. Unlike other restriction enzymes of its class, BglII has been shown to possess some unique structural characteristics, such as a Î²-sandwich subdomain, and appears to undergo a unique conformational change upon dimerization, but its overall structure and mechanism of catalysis remain consistent with other type II restriction enzymes.
Restriction endonucleases play a very important role in modern molecular cloning techniques. Because of their unique recognition/cut sites, restriction enzymes can be used to precisely cut DNA at specific locations in a predictable manner. Once cut, the DNA (usually) possesses so-called "sticky ends", which can then allow the DNA fragment to hybridise into a DNA vector. Ligating enzymes are used to covalently link the desired fragment to the vector for subsequent DNA cloning.
|Name||BglII Restriction Endonuclease|
|This phosphoryl transfer occurs by a nucleophilic attack of a hydride ion on the scissile phosphate, resulting in a trigonal bipyramidal phosphorus intermediate. The phosphorus then gets substituted and the 3'-0- is kicked off as a leaving group.|
BglII catalyses phosphodiester bond cleavage at the DNA backbone through a phosphoryl transfer to water. Studies on the mechanism of restriction enzymes have revealed several general features that seem to be true in almost all cases, although the actual mechanism for each enzyme is most likely some variation of this general mechanism. This mechanism requires a base to generate the hydroxide ion from water, which will act as the nucleophile and attack the phosphorus in the phosphodiester bond. Also required is a Lewis acid to stabilize the extra negative charge of the pentacoordinated transition state phosphorus, as well as a general acid or metal ion that stabilizes the leaving group (3â€™-Oâˆ’).
Although restriction endonucleases show little sequence similarity, crystal structures reveal that they all share a highly similar Î±/Î² core consisting of a six-stranded Î²-sheet flanked by five Î±-helices, two of which mediate dimerization. This core carries the active site (catalytic center) and the residues that contact DNA in the major groove. BglII is unique in that its Î±/Î² core is augmented by a Î²-sandwich subdomain that has several projections that extend outward to grip the DNA, allowing BglII to completely encircle the DNA molecule. This atypical feature of BglII suggests a unique hinge motion for DNA binding and release. Comparative structural studies of the free enzyme vs. the BglII-DNA complex showed that the enzyme opens by a dramatic scissor-like motion, accompanied by a complete rearrangement of the Î±-helices at the dimer interface. These structural studies also revealed that within each monomer a set of residues lowers or raises to alternatively sequester or expose the active site residues. These dramatic differences in structure in the free vs. bound enzyme have yet to be observed in any other restriction endonuclease and may possibly represent a novel mechanism for capturing DNA that may extend to other proteins that encircle DNA.
Structural studies of endonucleases have revealed a similar architecture for the active site with the residues following the weak consensus sequence Glu/Asp-(X)9-20-Glu/Asp/Ser-X-Lys/Glu. BglII's active site is similar to other endonucleases', following the sequence Asp-(X)9-Glu-X-Gln. In its active site there sits a divalent metal cation, most likely Mg2+, that interacts with Asp-84, Val-94, a phosphoryl oxygen, and three water molecules. One of these water molecules, is able act as a nucleophile because of its proximity to the scissile phosphoryl (its orientation being fixed by a hydrogen bond with the side chain amide oxygen of Gln-95) and its contact with the metal cation (which lowers its pKa, promoting the water's nucleophilicity).
- BamHI, a nuclease enzyme from 'Bacillus amyloliquefaciens..
- FokI, a nuclease enzyme from Flavobacterium okeanokoites
- EcoRI, a nuclease enzyme from 'E. coli.
- Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK (February 2000). "Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution". Nature Structural Biology. 7 (2): 134â€“40. doi:10.1038/72405. PMID 10655616.
- Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK (February 2001). "Structure of free BglII reveals an unprecedented scissor-like motion for opening an endonuclease". Nature Structural Biology. 8 (2): 126â€“30. doi:10.1038/84111. PMID 11175900.
- Galburt EA, Stoddard BL (February 2000). "Restriction endonucleases: one of these things is not like the others". Nature Structural Biology. 7 (2): 89â€“91. doi:10.1038/72450. PMID 10655603.
- Pingoud A, Jeltsch A (September 2001). "Structure and function of type II restriction endonucleases". Nucleic Acids Research. 29 (18): 3705â€“27. doi:10.1093/nar/29.18.3705. PMC 55916. PMID 11557805.
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Restriction endonuclease BglII Provide feedback
Members of this family are predominantly found in prokaryotic restriction endonuclease BglII, and adopt a structure consisting of an alpha/beta core containing a six-stranded beta-sheet surrounded by five alpha-helices, two of which are involved in homodimerisation of the endonuclease. They recognise the double-stranded DNA sequence AGATCT and cleave after A-1, resulting in specific double-stranded fragments with terminal 5'-phosphates .
Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK; , Nat Struct Biol. 2000;7:134-140.: Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution. PUBMED:10655616 EPMC:10655616
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR015278
Type II restriction endonucleases ( EC ) are components of prokaryotic DNA restriction-modification mechanisms that protect the organism against invading foreign DNA. These site-specific deoxyribonucleases catalyse the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. Of the 3000 restriction endonucleases that have been characterised, most are homodimeric or tetrameric enzymes that cleave target DNA at sequence-specific sites close to the recognition site. For homodimeric enzymes, the recognition site is usually a palindromic sequence 4-8 bp in length. Most enzymes require magnesium ions as a cofactor for catalysis. Although they can vary in their mode of recognition, many restriction endonucleases share a similar structural core comprising four beta-strands and one alpha-helix, as well as a similar mechanism of cleavage, suggesting a common ancestral origin [ PUBMED:15770420 ]. However, there is still considerable diversity amongst restriction endonucleases [ PUBMED:14576294 , PUBMED:11827971 ]. The target site recognition process triggers large conformational changes of the enzyme and the target DNA, leading to the activation of the catalytic centres. Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding as well, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone [ PUBMED:11557805 ].
There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements [ PUBMED:15121719 , PUBMED:12665693 ], as summarised below:
- Type I enzymes ( EC ) cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase ( EC ) activities.
- Type II enzymes ( EC ) cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase.
- Type III enzymes ( EC ) cleave at sites a short distance from recognition site; require ATP (but doesn't hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exists as part of a complex with a modification methylase methylase ( EC ).
- Type IV enzymes target methylated DNA.
This entry represents BglII restriction endonucleases, which recognise AGATCT and cleaves after A-1 [ PUBMED:10655616 , PUBMED:11175900 ]. BglII adopts a structure consisting of an alpha/beta core containing a six-stranded beta-sheet surrounded by five alpha-helices, two of which are involved in homodimerisation of the endonuclease.
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
|Molecular function||type II site-specific deoxyribonuclease activity (GO:0009036)|
|Biological process||DNA restriction-modification system (GO:0009307)|
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This clan includes a large number of nuclease families related to holliday junction resolvases [1,2].
The clan contains the following 145 members:AHJR-like ArenaCapSnatch BamHI BpuJI_N BpuSI_N Bse634I BsuBI_PstI_RE Cas_APE2256 Cas_Cas02710 Cas_Cas4 Cas_Csm6 Cas_DxTHG Cas_NE0113 CdiA_C CdiA_C_tRNase CoiA Csa1 Dna2 DpnI DpnII DpnII-MboI DUF1780 DUF1887 DUF2034 DUF2161 DUF234 DUF2357 DUF2726 DUF2800 DUF2887 DUF3799 DUF3883 DUF4143 DUF4263 DUF4420 DUF559 DUF5614 DUF6035 DUF6293 EC042_2821 EcoRI EcoRII-C eIF-3_zeta Endonuc-BglII Endonuc-BsobI Endonuc-EcoRV Endonuc-FokI_C Endonuc-HincII Endonuc-MspI Endonuc-PvuII Endonuc_BglI Endonuc_Holl ERCC4 Exo5 Flu_PA Herpes_UL24 Hjc HSDR_N HSDR_N_2 L_protein_N McrBC MepB MmcB-like Mrr_cat Mrr_cat_2 MTES_1575 MutH MvaI_BcnI NaeI NERD NgoMIV_restric NotI NucS PDCD9 PDDEXK_1 PDDEXK_10 PDDEXK_2 PDDEXK_3 PDDEXK_4 PDDEXK_5 PDDEXK_7 PDDEXK_9 Pet127 Phage_endo_I PND R-HINP1I Rad10 RAI1 RAP RE_AlwI RE_ApaLI RE_Bpu10I RE_BsaWI RE_Bsp6I RE_CfrBI RE_Eco47II RE_EcoO109I RE_endonuc RE_HaeII RE_HindIII RE_HindVP RE_HpaII RE_LlaJI RE_LlaMI RE_MjaI RE_NgoBV RE_NgoPII RE_SacI RE_ScaI RE_SinI RE_TaqI RE_TdeIII RE_XamI RE_XcyI RecC_C RecU RestrictionMunI RestrictionSfiI RmuC RNA_pol_Rpb5_N Sen15 SfsA Spo0A_C TBPIP_N ThaI Tn7_Tnp_TnsA_N Tox-REase-2 Tox-REase-3 Tox-REase-5 Tox-REase-7 Tox-REase-9 Transposase_31 tRNA_int_endo Tsp45I Uma2 UPF0102 Viral_alk_exo VirArc_Nuclease VRR_NUC Vsr XhoI XisH YaeQ YhcG_C YqaJ
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|Number in seed:||7|
|Number in full:||227|
|Average length of the domain:||184.80 aa|
|Average identity of full alignment:||28 %|
|Average coverage of the sequence by the domain:||83.68 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 57096847 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||13|
|Download:||download the raw HMM for this family|
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For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Endonuc-BglII domain has been found. There are 5 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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