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The restriction endonuclease BglII

Leo Laub '09


I. Introduction to Restriction Endonucleases

Restriction endonucleases are enzymes that can cleave specific sequences of double stranded DNA by cutting through the sugar-phosphate backbone of each strand. Currently, the exact mechanism behind this nucleolytic activity is unknown. However, in bacterial cells, they play an important role in the protection of the host genome from bacteriophage infection. Specifically, they target invasive genetic material for cleavage, preventing it from becoming incorporated into the host genome (Pingoud and Jeltsch, 1997).

Given they are involved in nucleolytic activity, it is vital that restriction endonucleases can target particular sequences for cleavage with exquisite specificity to avoid damaging endogenous genetic material. Restriction endonucleases accomplish this by binding non-specifically to DNA and sliding up or down it until reaching the target sequence. Cleavage only occurs if the target sequence is a perfect match; one nucleotide difference reduces nucleolytic activity by over one million-fold (Lukacs et al. 2000).

Scientists have categorized restriction endonuclases into three groups: type I, II, and III. The most studied group is type II, with more than 3,000 restriction endonucleases already identified. The orthodox type II endonucleases are characterized as those that recognize and cleave palindromic sequences of 4-8 bp in the presence of Mg2+. Unlike type I and III restriction endonucleases, the type II enzymes do not require ATP hydrolysis for full functionality. A variety of unorthodox type II restriction endonucleases also exist and are grouped into separate type II subgroups (Pingoud and Jeltsch, 2001).

II. General Structure of BglII and Interactions with DNA

BglII is a type II restriction endonuclease of 223 amino acids that targets the sequence AGATCT by encircling DNA from the major groove side (Lukacs et al. 2001). It is composed of two identical monomers that are bound together by two pairs of a-helices . This binding is mediated by hydrogen bonds between Asn98 residues on each of the a4 helices as well as by hydrophobic interactions between Pro 100, Leu 103, and Val 107, residues on each monomer . Each pair is part of a larger a/ß core composed of six alpha-helices and five beta-sheets . This core contains two important loops (B and C ) that contact bases in the DNA major groove.

Below the a/ß core of each monomer exists a ß-sandwich subdomain that interacts with the major and minor grooves of the DNA . This occurs via two loops: loop A interacts with bases in the DNA major groove while loop D interacts with the bases in the DNA minor groove. Additionally, loop D mediates the binding of BglII to DNA from the major groove side by encircling the double helix via hydrogen bonding between two tyrosine residues (Tyr 190) and a molecule of water (Lukacs et al. 2000). Upon binding, the DNA bends ~23° away from the protein .

III. Recognition of the BglII Consensus Sequence

A variety of chemical interactions mediate the specificity of BglII for its target sequence, GGATCC. As mentioned previously, four loops (A, B, C, and D) are responsible for protein-base pair interactions. However, only loops B, D, and C allow BglII to recognize its target sequence .

Loop B contains two residues that make sequence specific DNA contacts. Specifically, Ser 97 contacts the adenine of a T:A base pair via hydrogen bonding while Asn 98 interacts with the guanine of a G:C base pair via hydrogen bonding through a water molecule .

Loop D also contains two residues that make sequence specific interactions with the DNA. Arg 192 interacts with the thymine and adenine of an A:T base pair as well as with the guanine of a C:G base pair via hydrogen bonding mediated by two water molecules . Alternatively, a pair of Tyr 190 residues from each dimer coordinate hydrogen bonds through one water molecule to interact with two thymines from adjacent T:A base pairs .

The last two residues that allow the protein to detect specific sequences of DNA are attached to loop C. Ser 141 recognizes an A:T base pair by binding to an adenine via bidentate hydrogen bonds. Asn 140 recognizes the same base pair by forming a hydrogen bond with the associated thymine. Asn 140 also binds a cytosine bound to the guanine associated with Asn 98 (Lukacs et al. 2000).

IV. The BglII Active Site

After identifying the correct consensus sequence, BglII cleaves the phosophdiester bond between the adenine and guainine bases (A|GATCT), yielding 3'-OH and 5'-phosphate ends. Unfortunately, the exact process by which this occurs in BglII is currently unclear. Typically, however, the catalytic activity of restriction endonucleases occur via either an associative or dissociative mechanism.

The associative mechanism involves a base that generates a hydroxide ion to function as a nucleophile. The hydroxide ion attacks the phosophdiester bond at the phosphorus atom, causing the dissociation of the leaving group. To reach the transition state, in which the phosophorus atom is partially bound by five substituents, a Lewis acid must be available to stabilize the resultant negative charge on the phosphorus. An acid must also be present to allow dissociation of the leaving group via a protonation step.

The dissociative mechanism operates in much the same way, however, it does not require a base to produce a strong nucleophile. Instead, a strong base is necessary to stabilize the leaving group (Pingoud and Jeltsch, 2001).

While the precise mechanism by which BglII hydrolyzes the phosphodiester bond of DNA is unknown, more has been determined regarding the structure of its active site. Specifically, some metal cofactor sits in a position that allows interactions with Asp 84, Val 94, a phosphate oxygen, and three molecules of water. One of these water molecules likely functions as the nucleophile in the hydrolysis reaction, given its optimal location relative to the phosodiester bond . The catalytic center is further supported by the residues Asn 69, Glu 93, and Gln 95 (Lukacs et al. 2000).

While the identity of the metal cofactor has yet to be determined, homologous endonucleases are typically occupied by one or more divalent cations, such as Mg2+ (Pingoud and Jeltsch, 2001).

V. Structure of the Free Enzyme and a Mechanism for DNA Binding

A variety of conformations changes occur that allow free BglII to encircle DNA. Overall, though, these changes can be described as a "scissor-like" motion, in which the monomers of the enzyme spread apart by as much as ~50° to capture and cleave the correct target sequence of double stranded DNA ( versus ) (Lukacs et al. 2001).

The beginnings of such a conformational change likely occur when the protein encounters double stranded DNA. At such a point, BglII undergoes minimal structural changes that allow it to run along the DNA nonspecifically until it finds the consensus sequence. Only then does complete this "scissor-like" motion.

Many structural differences have been noted between the free protein and the DNA-bounded protein that has completed the "scissor-like" rotation of monomers. One of the most obvious is a change in orientation of what has been described as the "lever" (Asn 69-Asp 84) . When the enzyme is free from DNA, the "lever" is forced into the "down" position, hiding the active site from the DNA . Alternatively, when BglII has successfully bound its target sequence, the "lever" rotates to an "up" position, leaving the active site exposed for catalytic activity (Lukas, et al 2001).

Although lever movement inhibits the activity of the active site via steric hindrance, it also reduces such by promoting the formation of sequestering intramolecular bonds. Specifically, the catalytically active residues Asn 69, Asp 84, Glu 93, and Gln 95 are rendered useless by hydrogen bonding to Asn 105 and Arg 108 . Once complexed with DNA, however, such hydrogen bonds break and the active site regains its catalytic potential.

Binding by DNA also causes conformal changes in the orientation of helices associated with homodimerization (a4 and a5). Specifically, the a4 helix shortens by roughly one turn and shifts towards the a5 helix, precluding the symmetrically equivalent a4 and a5 helices of each dimer from interacting ( versus ).

Finally, other major differences between the two forms of the protein can be found by examining loops A, D, and E. In the free enzyme, these DNA contact motifs appear disordered. Once the protein has bound DNA, the loops become more structured and rigid.

VI. References

Aggarwal, A.K. Structure and function of restriction endonucleases. Current Opinion in Structural Biology. 1995. 5:11-19.

Lukacs, C., Kucera, R., Schildkraut, I., Aggarwal, A.K. Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution. Nature Sructural Biology. 2000. 2:134-140.

Lukacs, C., Kucera, R., Schildkraut, I., Aggarwal, A.K. Structure of free BglII reveals an unprecedented scissor-like motion for opening an endonuclease. Nature Sructural Biology. 2001. 8:126-130.

Pingoud, A., Jeltsch, A. Recognition and leavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 1997. 246:1-22.

Pingoud, A., Jeltsch, A. Structure and function of type II restriction endonucleases. Nucleic Acids Research. 2001. 29:3705-3727.

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