restriction endonuclease

RESTRICTION ENDONUCLEASESBglII&BamHI : 

RESTRICTION ENDONUCLEASESBglII&BamHI Mrs.P.Sugapriya Menaga

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Introduction: RE are paradigms for study of the Protein-DNA recognition. Type II RE have served as a models for understanding the basis for site specific DNA recognition & cleavage by proteins. Form homodimers,depend on Mg 2+ activity,&cleave within a palindrome recognition sequence of 4-8 bp. Crystal structures of Type II RE such as BamHI, BglII, EcoRV revealed a structural basis for specific protein-DNA complex formation,nonspecific association with DNA & catalytic activity of these enzymes. Lukacs et.al provide a analysis of endonuclease specificity by comparing the structure of BglII to the BamHI. They display different protein-DNA contacts at common target site bp.They related by common ancestry ,evolved independent mechanisms for both DNA binding & cleavage. The structure of BamHI-DNA complex reveals that the enzyme recognizes its cognate sequence GGATCC primarily through Asn116, Ser118, Arg122, Asp154.

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Endonuclease BamhI Protein_DNA complex

Structure of BglII : 

Structure of BglII Crystal structure of BglII complexed with DNA 16 mer. X-ray diffraction I.70 Angstroms

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BglII: BglII,a 223 aa protein ,isolated from B.globigii & its sequence shows little homology to other endonucleases,including BamHI. The enzyme was cocrystalised with 16-bp DNA fragment containing its recognition sequence AGATCT.The structure was determined at 1.5 Å. The lack of sequence homology between BglII & BamHI, the two enzymes share a similar / core. In BglII, the / core is augmented by a -sandwich sub domain that provides additional specificity. The common bp AGATCT are recognized in different ways in two complexes because of the differences in DNA conformation.

BglII-DNA : 

BglII-DNA At 1.5 resolution,the BglII-DNA structure provides detailed picture of the BglII-DNA recognition. BglII dimmer approaches DNA from the major groove side & wraps around to the minor groove side.The monomer is composed of two domains: an / core domain that bears resemblance to BamHI, & a unique five stranded -sandwich domain. Loops from the -sandwich domain reach into the both the major & minor grooves to tightly grip the DNA .

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The BgLII-DNA& BamHI-DNA complex rotated 90.The / Core is a gray molecular surface & rest of proteins in ribbon form. BglII Asp38, Tyr190, &Arg189 from loop A&D of the - sandwich & a water mol participate in H- bond encloses the DNA.BamHI has small secondary domain.

BglII : 

BglII BglII-DNA complex looking down the DNA axis.  helices,  strands & loops A-E are labeled on one monomer. loops B & C make direct DNA contacts. loops D & A involved in water- mediated contacts.

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PROTEIN CONFORMATION: The / cores similar to that of the BamHI,containing a six-stranded  sheet(1,3,4,5,6 & 7) surrounded by five  helices(1,2,3,4 & 5), two of which (4 & 5) are involved in homodimerization. The loops preceding the dimerization helices(loops B & C) carry the residues that contact bases in the major groove. The BglII & BamHI cores differ in the lengths of the  helices &  strands. BamHI has a small two-stranded -substructure outside of the / cores. In BglII ,this substructure is elaborated into a full-five stranded -sandwich domain (2, 8,9,10 & 11) that extends the overall size & shape of the protein. 2 is composed of residues near the N-terminus ,the rest of the domain composed of the C-terminal 50 residues. 2 form a long loop(A) that project into the DNA major groove. The loop between 8 & 9 (loopD) is the site for interaction bases in the minor groove.

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The loop between 10 & 11(loopE) is visible only one of the monomers. The second sub domain gives the BglII-DNA complex a “square like” appearance when view down the DNA helix. DNA CONFORMATION: DNA duplexes form continuous,sinuous helices through out crystal. The BglII rs (AGATCT) is unwound by ~15 as compared to the BamHI complex. Overwinding by ~7 between the inner & middle bp (AGATCT). This compensates for the unwinding at the center. The outer recognition bp (AGATCT) in the BglII-DNA are only a few  offset from the equivalent bp in BamHI(GGATCC) complex. DNA RECOGNITION: The BglII recognition site differs from that of BamHI by only the outer bp. BamHI --- GGATCC CCTAGG BglII --- AGATCT TCTAGA

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Asn116,recognizes the inner & middle bp in BamHI(GGA) has an equivalent residue in BglII(Asn98) but it adopts a different configuration. Super imposition of the two enzymes shows that Asn98 is unable to interact with DNA as Asn116 in BamHI, as a result bending & other helical parameters between the two DNA. Asn98 makes water-mediated hydrogen bond to middle G. No direct contacts to the inner bp A:T in BamHI,but in BglII a direct H-bond is formed b/w the N7 of A & O of Ser 97 from the two-fold monomer. Common interaction b/w BamHI &BglII is a H-bond b/w the middle G:C bp & the main chain carbonyl of Asn140(Asn154 in BamHI). Asn140 & Ser141 in BglII substitute for Asp154 & Arg155 in BamHI to recognize an A:T bp in place of G:C bp. In BamHI, Arg155 donates bidentate H-bonds to the outer G while Asp154 accepts H-bond form C. In BglII, Ser141 forms bidentate H-bonds to the outer A,& Asn140 donates single H-bond to the O4 of T. In BamHI minor groove contacted asymmetrically by a C-terminal arm from one subunit, but in BglII symmetrically by a loopD from the -sandwich, which wraps around minor groove to make water-mediated contacts with all 3 bp in each half-site.

Secondary Structure of BglII : 

Secondary Structure of BglII Main-chain trace Chain: A ( 223 residues ) – [6 helices, 11 strands ] Chain: B ( 218 residues ) – [7 helices, 11 strands ] Nucleic acids Chain: C ( 16 bases ) Chain: D ( 16 bases)

Secondary structure of BamHI : 

Secondary structure of BamHI Main-chain trace Chain: A ( 198 residues ) – [9 helices, 6 strands ] Chain: B ( 208 residues ) - [9 helices, 8 strands ] Nucleic acids Chain: C ( 12 bases ) Chain: D ( 11 bases )

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ACTIVE SITE: BamHI contains the a glutamate(Glu113) at the final position of the consensus. Mutating this residue to lysine inactivates the enzyme. BglII active site is similar to other endonucleases but follows the sequence Asp 84-X-Glu 93-X-Gln 95. BamHI becomes inactive when Glu113 is mutated to a glutamine as in BglII The presence of a single metal in the BglII active site contrasts with BamHI ,which contains two metals in the active site,are involved in the stabilization the pentacovalent transition state

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Ribbon diagrams of protein-Dna complexes of BglII & I-CreI.

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BglII & BamHI share a / core that carries the catalytic center & residues that contact DNA bases in the major groove. The protein-DNA contacts are different from both. Based on the similarity of their folds , BglII & BamHI diverged from common ancestors.both mutational & structural data indicate that they have diverged to the point where chemical differences in the active site & structural differences in DNA binding preclude the possibility of altering specificity by simple mutagenic events.