Type
II Restriction Endonuclease HinP1I
Aaron Yeoh '12 and Nathan Huey '13
Contents:
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
Back to Top