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Type II Restriction Endonuclease HinP1I

Aaron Yeoh '12 and Nathan Huey '13


I. Introduction

Restriction endonucleases are nucleolytic enzymes that are capable of cleaving specific sequences of double stranded DNA by making cuts at the phosphate-sugar backbone. Restriction enzymes are expressed ubiquitously throughout prokaryotic organisms. Their primary biological function is to protect the host genome against invading exogenous DNA such as threatening bacteriophage DNA. Other functions of restriction enzymes are still being investigated, such as a role in homologous recombination and transposition of DNA (Pingoud and Jeltsch, 2001).   

Restriction endonucleases have been classified according to their subunit composition, required metal cofactor and mechanism of action, type I, II or III (Pingoud and Jeltsch, 1997).   Over 3600 Type II restriction endonucleases have been categorized, possessing more than 250 different specific DNA sequences. Unlike Type I and III restriction endonucleases, which recognize asymmetric DNA sequences, type II enzymes recognize palindromic DNA of 4-8 bp (Yang et al. 2005). Type II restriction endonucleases interact with DNA in a complex manner. First, the enzyme binds non-specifically to DNA and then slides by random diffusion until coming into contact with its recognition sequence. Then the enzyme undergoes a conformational change, leading to activation of the catalytic site. Following phophodiester bond cleavage, the enzyme leaves by either direct dissociation or transfer-enzyme mediated departure (Pingoud and Jeltsch, 2001).

Type II restriction enzymes are of particular interest to molecular biologists due to their importance in genetic manipulation. Furthermore, understanding the protein/DNA interactions of restriction endonucleases provide valuable insights into the structure-function relationships that drive molecular processes (Pingoud and Jeltsch, 2001).

II. General Structure and Binding with Cognate DNA

HinP1l is a Type II restriction endonuclease from the gram-negative bacteria Haemophilus influenzae. HinP1l recognizes and cleaves the palindromic DNA sequence G↓CGC producing 2 nucleotide overhangs at the 5’ ends (Horton et al. 2005).

HinP1l is comprised of 247 amino acids and belongs to the α/β protein class. The protein contains two sets of β-sheets surrounded by eight α-helices (αA- αH). Four of the helices sandwich the upper β-sheet, which is six stranded and mixed,  with some strands parallel and others antiparallel. The lower β-sheet is made up of five antiparallel strands and is packed on one side by 5 α-helices , the other side forming a concave, basic DNA binding surface. Two structures involved in DNA binding are the long, 7-turn αA helix, binding in the minor groove, and the β-strands with their associated loops, binding the major groove .

Unlike most Type II restriction endonucleases, each HinP1I monomer binds a DNA duplex. Numerous direct and water mediated interactions are made between the HinP1I monomer and the phosphate backbone of the DNA. In addition to these stabilizing interactions, the bases of the recognition sequence H-bond directly with the monomer. The four guanines interact through their O6 atoms with the side chains of K96, K223, K238, and Q236 . All four cytosines H-bond via their N4 atoms with oxygen atoms from two side chains (D226 and Q93) and two main chain carbonyl oxygen atoms (K223 and F91) .

Upon binding to a cognate DNA sequence the αA helix is extended in the N-terminal direction for 17 residues. In the unbound form the first 6 residues are invisible in the electron density map, residues 9 through 11 are in a β-strand, and residues 12-17 are in a loop connected to the N-terminal helix . The extended helix then clamps down upon the minor groove of the DNA. This causes the DNA around the two central base pairs in the recognition sequence to adopt an A-form conformation, resulting in a wider minor groove . In contrast, the β-strands show very little change upon DNA binding, indicating that the catalytic site is pre-formed and rigid.  

III. DNA Distortions (Bending and Base-Flipping)

Two major changes to DNA structure occur at the ends of the recognition sequence upon binding to HinP1I . On one side, the DNA is bent approximately 60 degrees by the intercalation of the hydrophobic phenyl group of F91 from the major groove . On the other end of the recognition sequence, the guanine interacts in the minor groove through van der Waals contact with F15 from the N-terminal helix . Interestingly, in some cases, a base was found to flip out from the regular helical pattern of the DNA. This flipped base was stabilized by residues H97, W98, M234 as well as a  5’ cytosine base . The local DNA structure adopts a Z-DNA conformation. The physiological relevance of this mechanism is unknown, but is the first documented example of a nucleotide outside of a recognition sequence that is only flipped out under certain conditions.

IV. Catalytic Site

Two divalent magnesium ions are needed to bind as ligands for the cleavage reaction to occur . The first magnesium is bound in an octahedral manner to the side chain oxygen atoms of D62 and Q81, the main chain carbonyl atom of V82, the oxygen O1P of the scissile phosphate and a water molecule (w1). This water acts as the nucleophile in the reaction that cleaves the DNA and also interacts with the O1P oxygen of the 3’ scissile phosphate and the O2P oxygen of the 3’ nucleotide through H-bonding. The second metal is bound by side chain oxygen of D62 and O2P, the leaving group O3’ oxygen of 5’ guanine, the oxygen O1P of the scissile phosphate, and a second water molecule (w2) . The catalytic site of HinP1I contains a motif similar to the catalytic motif of MutH , but with the major replacement of glutamate 77 of MutH for glutamine 81 of HinP1I. A nitrogen atom of the side chain of Q81 interacts with the main chain carbonyl oxygen of K60, possibly adding stability to the active site .

The K83 residue is essential to the function of HinP1I, linking sequence-specificity with ligand binding and the resulting cleavage. If DNA is bound without the metal ions, the amino group of K83 is H-bonded with the carbonyl oxygen of Q93, a base-recognition residue . When the metal ion is bound, it is instead H-bound to the side chain oxygen of Q81, a metal coordinator . Finally, after DNA cleavage the amino group is within H-bonding distance of both of these residues as well as the scissile phosphate group .

V. Cleavage Mechanism and Unique Dimerization

HinP1I has been found to dimerize with a major part of the dimer interface formed by the α-helices αG and αF . The interactions between the parallel four-helix bundle that forms include van der Waals bonding, H-bonding, and electrostatic interactions. Residue R166 interacts with E174 and N194 while R168 interacts with E195 .

Interestingly, the catalytic sites of the dimer face out from each other, rather than coming together to form the core of the dimer. With most restriction endonucleases each strand of a DNA duplex can be bound and cut simultaneously. However, the duplex of DNA binds to a single monomer of HinP1I. Horton et al (2005) treated supercoiled plasmids with HinP1I, EcoRI (an enzyme that is known to cleave both strands), or BamHI (cleaves strands by nicking strands sequentially). They found that HinP1I causes a nicked product to accumulate before full cleavage. It has been suggested that the enzyme may stay in the vicinity of the nicked DNA, binding the opposite strand with high probability and completing the cleavage of dsDNA (Horton et al, 2005).

VI. References

Horton, J. R., Zhang, X., Maunus, R., Yang, Z., Wilson, G. G., Roberts, R. J., & Cheng, X.DNA nicking by HinP1I endonuclease: Bending, base flipping and minor groove expansion. Nucleic Acids Research, 34(3), 939-948. doi:10.1093/nar/gkj484

Pingoud, A., & Jeltsch, A. (1997). Recognition and cleavage of DNA by type-II restriction endonucleases. European Journal of Biochemistry, 246(1), 1-22.

Pingoud, A., & Jeltsch, A. (2001). Structure and function of type II restriction endonucleases. Nucleic Acids Research, 29(18), 3705-3727. doi:10.1093/nar/29.18.3705

Yang, Z., Horton, J. R., Maunus, R., Wilson, G. G., Roberts, R. J., & Cheng, X.Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI. Nucleic Acids Research, 33(6), 1892-1901. doi:10.1093/nar/gki337

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