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9  structures 13  species 1  interaction 15  sequences 2  architectures

Family: BamHI (PF02923)

Summary: Restriction endonuclease BamHI

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BamHI Edit Wikipedia article

BamH I
PDB 1esg EBI.jpg
Restriction endonuclease BamH I bound to a non-specific DNA.
Identifiers
Symbol BamH I
Pfam PF02923
Pfam clan CL0236
InterPro IPR004194
SCOP 1bhm
SUPERFAMILY 1bhm

BamH I (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 b.p.) of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamH I as described by Newman, et al. (1995). BamH I binds at the recognition sequence 5'-GGATCC-3', and cleaves these sequences just after the 5'-guanine on each strand. This cleavage results in sticky ends which are 4 b.p. long. In its unbound form, BamH I displays a central b sheet, which resides in between a helices. BamH I is an extraordinarily unique molecule in that it undergoes a series of unconventional conformational changes upon DNA recognition. This allows the DNA to maintain its normal B-DNA conformation without distorting to facilitate enzyme binding. BamH I is a symmetric dimer. DNA is bound in a large cleft that is formed between dimers; the enzyme binds in a "crossover" manner. Each BamH I subunit makes the majority of its backbone contacts with the phosphates of a DNA half site but base pair contacts are made between each BamH I subunit and nitrogenous bases in the major groove of the opposite DNA half site. The protein binds the bases through either direct hydrogen bonds or water-mediated H-bonds between the protein and every H-bond donor/acceptor group in the major groove. Major groove contacts are formed by atoms residing on the amino-terminus of a parallel 4 helix bundle. This bundle marks the BamH I dimer interface, and it is thought that the dipole moments of the NH2-terminal atoms on this bundle may contribute to electrostatic stabilization.

Sites of Recognition Between BamH I and DNA

The BamH I enzyme is capable of making a large number of contacts with DNA. Water-mediated hydrogen bonding, as well as both main-chain and side-chain interactions aid in binding of the BamH I recognition sequence. In the major groove, the majority of enzyme/DNA contacts take place at the amino terminus of the parallel-4-helix bundle, made up of a4 and a6 from each subunit. Although a6 from each subunit does not enter the DNA major groove, its preceding loops interact with the outer ends of the recognition site. Conversely, a4 from each subunit does enter the major groove in the center of the recognition sequence. A total of 18 bonds are formed between the enzyme and DNA across the 6 base pair recognition sequence (12 direct and 6 water mediated bonds). As discussed above, the L and R subunits bind in a cross over manner, whereby the R-subunit of BamH I contacts the left DNA half-site of the recognition sequence. The binding of each BamH I subunit is precisely the same as its symmetrical partner. The recognition site for BamH I has a palindromic sequence which can be cut in half for ease in showing bonds.

Recognition site

G G A T C C
C C T A G G

As of the end of 2010, there were 5 crystal structures of BamH I in the Protein Data Bank

Two-metal Mechanism

BamHI, type II restriction endonucleases, often requires divalent metals as cofactors to catalyze DNA cleavage.[1] Two-metal ion mechanism is one of the possible catalytic mechanisms of BamHI since the BamHI crystal structure has the ability to bind two metal ions at the active site, which is suitable for the classical two-metal ion mechanism to proceed. Two-metal ion mechanism is the use of two metal ions to catalyze the cleavage reaction of restriction enzyme. BamHI has three critical active site residues that are important for metal catalyst. They are known as Asp94, Glu111 and Glu113. These residues are usually acidic. In the presence of a metal ion, the residues are pointed toward the metal ion. In the absence of metal ions, the residues are pointed outward. The two metal ions (A and B) are 4.1 apart from each other in the active site and are in-line with these residues.[2] In general, when the two metal ions (A and B) are bonded to the active site, they help stabilize a cluster distribution of negative charges localized at the active site created by the leaving of an oxygen atom during the transition state. First, a water molecule will be activated by metal ion A at the active site. This water molecule will act as the attacking molecule attacking the BamHI-DNA complex and thus making the complex negative. Later, another water will bound to metal ion B and donate a proton to the leaving group of complex, stabilizing the build-up of negative charge on the leaving oxygen atom.[3]

The function of Ca2+ in the active site of BamHI is known. It is an inhibitor of DNA cleavage, converting BamHI into the pre-reactive state. This revealed the water molecular is the attacking molecule. It donates a proton to the leaving group that is bounded to Ca2+ forming a 90o O-P-O bond angles. If Glu 113 is replaced by lysine, the cleavage is lost since Glu 113 accepts the proton from the attacking water molecule.[2]

References

  1. ^ Ninfa, Alexander (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Wiley. p. 345. ISBN 978-0470087664. 
  2. ^ a b Viadiu, hector (1998). "The role of metals in catalysis by the restriction endonuclease BamHI". Nature Structural Biology. 
  3. ^ Mordasini, Tiziana (December 18, 2002). "Why do divalent metal ions either promote or inhibit enzymatic reactions? - The case of BamHI restriction endonuclease from combined quantum-classical simulations". The Journal of Biological Chemistry. 
  • M. Newman, T. Strzelecka, F.D. Dorner, I. Schildkraut, A. K. Aggarwal, Science. 269, 656 (1995)

External links

This article incorporates text from the public domain Pfam and InterPro IPR004194

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Restriction endonuclease BamHI Provide feedback

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Literature references

  1. Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK; , Nature 1994;368:660-664.: Structure of restriction endonuclease BamHI and its relationship to EcoRI. PUBMED:8145855 EPMC:8145855


External database links

This tab holds annotation information from the InterPro database.

InterPro entry IPR004194

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.

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].

This entry represents BamHI restriction endonucleases, which recognises the DNA sequence GGATCC and cleaves after G-1 [PUBMED:8145855, PUBMED:10882125].

Domain organisation

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Seed source: Structural domain
Previous IDs: none
Type: Domain
Author: Griffiths-Jones SR
Number in seed: 4
Number in full: 15
Average length of the domain: 130.90 aa
Average identity of full alignment: 46 %
Average coverage of the sequence by the domain: 76.17 %

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HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 11927849 -E 1000 --cpu 4 HMM pfamseq
Model details:
Parameter Sequence Domain
Gathering cut-off 22.8 22.8
Trusted cut-off 30.1 29.5
Noise cut-off 21.2 20.1
Model length: 157
Family (HMM) version: 12
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Interactions

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BamHI

Structures

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 BamHI domain has been found. There are 9 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 seqence.

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