BamHI (pronounced "Bam H one") (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamHI as described by Newman, et al. (1995). BamHI 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 bp long. In its unbound form, BamHI displays a central b sheet, which resides in between α-helices.
BamHI 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. BamHI is a symmetric dimer. DNA is bound in a large cleft that is formed between dimers; the enzyme binds in a "crossover" manner. Each BamHI subunit makes the majority of its backbone contacts with the phosphates of a DNA half site but base pair contacts are made between each BamHI 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 BamHI dimer interface, and it is thought that the dipole moments of the NH2-terminal atoms on this bundle may contribute to electrostatic stabilization.
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Restriction enzymes
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AP Biology: Restriction Enzyme Digests on Circular Plasmids
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Restriction Enzymes (Restriction Endonucleases)
Transcription
- [Voiceover] All right, so in this video, we're gonna be talking about something known as restriction enzymes. Now, what are restriction enzymes? Well, let's go through an example, and hopefully that'll help answer that question. So let's imagine that we've got a bacteria. And it's just floating around, it's doing its thing, and out of nowhere comes this virus. And the virus is just kinda upset today, so it decides that it wants to infect this bacteria. So, what the virus does is it goes over to the bacteria, and basically it attaches to it, and it injects viral DNA into the bacteria. So this is the viral DNA, and I'll just label that here, so viral DNA. And the bacteria, being a living creature, also has its own DNA. So this right here in the blue, is the bacterial DNA, and I'll just label that here. So bacterial DNA. So basically, the virus infected the bacteria. But the bacteria wants to figure out some way to destroy the viral DNA. And one way that it can do that, is by labeling its own DNA. So let's imagine that the bacteria labels its DNA with these purple dots. So what these purple dots actually are, is a methyl group. So we'll say that these purple dots are methyl groups. And in order for the bacterial DNA to be methylated, there's an enzyme known as methylase. So methylase is an enzyme that basically floats around, and as bacterial DNA is synthesized, this enzyme methylase goes around and methylates bacterial DNA. Now, what this basically does, is it allows the bacteria to recognize its own DNA, and recognize any DNA that's not methylated as foreign DNA. So what we now have is another enzyme that's floating around in the cytoplasm of the bacteria. And that enzyme is known as a restriction enzyme. So this restriction enzyme. So this restriction enzyme is kind of floating around, it's doing its thing, and it recognizes the methylated bacterial DNA, but then it sees this foreign, unmethylated DNA, and it goes and destroys it. So this is basically a way for bacteria to protect itself from being infected by viruses. And it basically does so by methylating its own DNA, and destroying any other foreign DNA that is unmethylated. So the reason that this is called a restriction enzyme, is because researchers were noticing that certain bacteria were actually, they were resistant to being infected by viruses. So they were basically restricting viral growth. Thus, when they figured out what enzyme is causing that, they basically called it a restriction enzyme, 'cause it restricted the growth of viruses. Now, in order for the restriction enzyme to work, it needs to recognize a specific sequence, a specific bacterial sequence. So let's go ahead and write out an examaple sequence. So imagine that, if we zoomed down to the nucleotide sequence of the bacterial DNA, let's imagine that maybe the sequence was G-A-A-T-T-C. And of course, if this was one strand, then the sister strand would be G-A-A-T-T-C. Now, I don't know if you notice, but there's something unique about this specific sequence. So if you read this top line from left to right, G-A-A-T-T-C, and you read this bottom strand from right to left, G-A-A-T-T-C, they're basically identical. And this is known as a palindromic sequence. So palindromic sequence. Now, basically what a palindromic sequence is, is exactly this and it's what restriction enzymes recognize. So let's imagine that we have a restriction enzyme, we'll just give it a name, let's imagine that its name is EcoR1, so EcoR1. That's the name of one type of restriction enzyme. EcoR1 is actually able to recognize this palindromic sequence. So as EcoR1 is floating around the bacterial cell, it'll recognize this sequence, and if it's methylated, it won't touch it. But if it's unmethylated, as in the viral DNA, it'll actually cleave it, so it'll come here, and it'll cleave it. Let me actually use a different color. So it'll cleave the DNA. And it'll cut it like so. And what that'll do is it'll result in two strands. Because it just cut the DNA. So now what we're gonna have is we're gonna have one strand that's gonna be G, and then this bottom strand, T-T-A-A, and then we're gonna have this top part over here. So we're gonna have an A-A-T-T-C, and then a G down here. So basically, now these two strands are just floating around, and these ends down here, so this end kinda hanging over the edge, they're known as sticky ends. So these are known as sticky ends. And the sticky ends basically will float around, and if they contact one another, since they're complementary strands, they'll just reanneal. So normally if this gets cut, then you'll have two sticky ends, they'll float around and then they'll reanneal. And we can actually take advantage of the fact that we can have the strands reanneal, and we'll talk about different ways that we can actually use that for medicinal purposes. So one way that we can use this to our advantage, would be, let's say that we wanna synthesize human insulin. So let's say that I wanna make human insulin. So how can I make human insulin using the restriction enzyme technology? Well, let's imagine that we have a bacteria over here. And this bacteria has its bacterial DNA inside. And what we can basically do, is take this bacterial DNA, so let's extract it out of the cell, so we'll move it out here. So here's the bacterial DNA. What we'll do is we'll take EcoR1, and we'll basically cleave the bacterial DNA at this site. So let's say that we cut it right here. So if we cut it right there and then we kinda unroll the bacterial DNA, now we have this space. And this space basically has these sticky ends here. So I'll just fill those in. So let's say that there's a G C-T-T-A-A, and then over here on this side, I don't know if I have enough space, we'll have an A-A-T-T-C and a G. So now we've got these sticky ends over here. And what we can go ahead and do is we can take the human insulin gene. So we'll take human insulin, so this is the insulin gene. And we'll have exposed it to EcoR1 already. So now it's got the sticky ends over here. So now on this side we've got an A-A-T-T-C and a G, and then over here on this side we've got a G, and a C-T-T-A-A. So now what we can do is we can take this whole thing and just plop it right in here. And as you can see, this part of the insulin gene will reanneal with this right here. And then this part of the insulin gene will reanneal with this part of the bacterial DNA. And basically now what we have is we have the insulin gene inserted into the bacterial DNA. And what that'll basically allow the bacteria to do is it'll allow it to synthesize human insulin. And now what you do is you just take that insulin and you purify it, and now you can basically have a whole bunch of insulin that's made very cheaply and very quickly for diabetic patients that might need the insulin. So that's one example of how restriction enzymes can be used in the pharmaceutical and biotechnological world.
Sites of Recognition Between BamHI and DNA
The BamHI 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 BamHI 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). Arg155 and Asp154 located in a spiral ring before a6 are connected with G:C base pairs outside while the middle G:C pairs are connected with Asp154, Arg122, and Asn116 (direct binding). Hydrogen bonding between water and Asn116 results in binding at A:T base pairs inside (water-mediated binding).[1] 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, like other type II restriction endonucleases, often requires divalent metals as cofactors to catalyze DNA cleavage.[2] 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.[3] 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.[4]
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.[3]
Biological significance
Because of its ability to recognize specific DNA sequence and cleave by a nuclease, BamHI carries various importances in understanding Type II restriction endonuclease, cloning DNA, and possibly treating certain DNA mutation-derived diseases through genetic therapy.[1] NARP and MILS syndromes, for example, are mitochondrial diseases that can be caused by mutations in the mitochondrial DNA. Mitochondria can recover its functions after the excision of the mutant sequence through restriction endonuclease.[5]
References
- ^ a b Tong Z, Zhao B, Zhao G, Shang H, Guan Y (September 2014). "2'-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII". Journal of Biosciences. 39 (4): 621–30. doi:10.1007/s12038-014-9466-4. PMID 25116617. S2CID 15537179.
- ^ Ninfa AJ, Ballou DP, Benore M (2008). Fundamental Laboratory Approaches for Biochemistry and Biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p. 345. ISBN 978-0-470-08766-4.
- ^ a b Viadiu H, Aggarwal AK (October 1998). "The role of metals in catalysis by the restriction endonuclease BamHI". Nature Structural Biology. 5 (10): 910–6. doi:10.1038/2352. PMID 9783752. S2CID 25062657.
- ^ Mordasini T, Curioni A, Andreoni W (February 2003). "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. 278 (7): 4381–4. doi:10.1074/jbc.C200664200. PMID 12496295.
- ^ Alexeyev MF, Venediktova N, Pastukh V, Shokolenko I, Bonilla G, Wilson GL (April 2008). "Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes". Gene Therapy. 15 (7): 516–23. doi:10.1038/sj.gt.2008.11. PMID 18256697.
Further reading
- Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK (August 1995). "Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding". Science. 269 (5224): 656–63. Bibcode:1995Sci...269..656N. doi:10.1126/science.7624794. PMID 7624794.
External links
- Deoxyribonuclease+BamHI at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
- 5 crystal structures[dead link]
This page was last edited on 2 December 2023, at 19:09