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A]
RESTRICTION AND MODIFICATION OF DNA
28
B]
TYPE II RESTRICTION ENDONUCLEASES
28
(i)
(ii)
(iii)
C]
General Properties
Structure Of Restriction Endonucleases
(a) EcoR I
(b) EcoR V
Catalytic Mechanism
Literature Review on
BamH I
37
(A) RESTRICTION AND MODIFICATION OF DNA
Restriction
identified
involved
1968).
and
modification
of DNA
in
bacteria
were
first
over 30 years ago and the first characterization of an
in
this process was described 25 years ago
(Meselson
A R/M system must possess two enzyme activities,
the
enzyme
and
Yuan
restriction
endonuclease and the modification methylase, both of which are dependent on
recognition of
1982)
same
The restriction
sequence
so
the
DNA sequence (Bickle 1982; Modrich
activity cleaves the DNA, but only if
is not methylated.
there is no cleavage
barriers
Arber
Roberts
recognition
In the bacterium both system are active
of its own genome.
But this
mechanism
and
provides
against both interspecies genetic transfer and phage infection
1979).
Many R/M
genetic
analysis
reveals
three
system
have now been characterized
and subsequently
different
1987). For type
I
and
classes
III
of type I and two in the
i.e.,
type
trast the type II systems consist
fication and one for restriction
of type
I,
II
and
III
by
(Bickle
are functions of one
of different
case
initially
by purification of the proteins and it
both activities
meric protein made up of a number
case
and
oligo-
polypeptides, three in the
III (Bickle 1982).
In
of two separate proteins, one for
conmodi-
(Modrich and Roberts 1982).
\B) TYPE II RESTRICTION ENDONUCLEASES
(i) General properties
Type II restriction endonucleases are the simplest and the
common
(Roberts 1990).
teins.
prise
separate
The recognition sequences are essentially symmetric,
Cleavage
in
The
to
act mainly as
homodimers,
the
R and M genes occur in all linkage
28
The
com-
additional
interruptions (Wilson
occurs symmetrically within the sequences.
believed
monomers.
the form of non specific
pro-
they
four to eight specific nucleotides, but they may include
nucleotides
are
The endonuclease and methylase are
most
1991).
endonucleases
methyltransferases
configuration
as
(Wilson
1988).
Most
first;
at other
genes
often,
the genes are aligned; sometimes the R genes
times
the
comes
M genes come first. In several systems
have opposite orientations; some diverge, other
converge
the
(Wilson
1991).
A tremendous amount of diversity is seen
II restriction enzymes.
among the various Type
At present over 640 different type II
restriction
enzymes
have been identified from a wide variety of bacterial genera
between
them,
1987).
The
Nathans
nomenclature
given
by
(Roberts
Smith
the same DNA sequence.
which
each
usually
Most
position
1987).
Hha I, EcoRI
tion
sequence.
observation
But
more
specific for
(pu)
which the
specified
Moreover,
DNA sequences.
and pyrimidines (py).
for Cau I and
and
these
in
are
recogni-
For example, Hind II
sequence
sequences,
EcoRV provide examples of this type of
bases.
unique
to
(Roberts
quences in which certain position
tides.
either
other enzymes such as Hha II and
unspecified
found
4 or 6 bp or rarely 8bp
discontinuous sequences in
one or
unique
is fully-specified by a given base,
and
of
Two or more of such enzymes are known as
of these enzymes recognize
continuous sequences of
and
The discrepancy between the numbers
enzymes and recognition sequences arises from the
"isoschizomers".
Cau
sequences
restriction enzymes from different bacteria are frequently
recognize
are
DNA
widely accepted is that
(Smith and Nathans 1973).
restriction
that
they recognize over 135 different
and,
Sfi
recognize
bases- are interrupted by
not
all
restriction enzymes
Many recognize
can be
I,
degenerate
se-
occupied by alternative
nucleo-
can act upon various combinations of
purines
Second type is exemplified by the
Cau II.
recognition
The central position of DNA sequence
I is either A-T or T-A while for
Cau II its target sequence can
have
either G-C or C-G as the
central
Unique recognition sites
for restriction enzymes are nearly always symmet-
rical,
both
strands of the
sequences
are
number
type II
quences
of
called
base pair.
for
DNA having
palindromes.
restriction
(Bennett and Halford; 1989.)
the
same
However,
enzymes that
but at sites that have no symmetry
29
5'-3'
there
also exist a
recognize
at all;
sequence.
Mba
unique
I
Such
small
DNA
se-
provides
one
such example.
Degenerate
rical or asymmetrical,
either
recognition sequences
depending
on
I or Hind II).
both
strands of DNA within
be
either
by the degeneracy retain
The majority of type II restriction
their
symmet-
the nature of the degeneracy, but in
case the nucleotides unaffected
(Cau
may
recognition sequence
With some, the cleavage occurs in the centre of
symmetry
enzymes
cleave
(Roberts
1987).
the recognition site
(for
example, EcoR V or Hind II), and in these cases the reaction products
will
be double stranded DNA with flush termini.
cleaved
and
towards the 5' end
of
the
With others,
recognition sequence
Eco RI) or towards the 3' end (viz Hha
I),
products will be duplex DNA that carry single
5'
or
3' termini, respectively.
both
strands are
(example Cau
and here
their
I
reaction
stranded extensions at their
However, when two
or
more
restriction
enzymes cleave DNA at the same site, these isoschizomers do not necessarily
cleave
the
same
phosphodiester bonds.
For
example,
the
DNA
recognized
by Cau II is also the target for both Nci I and
while
I cuts the same bonds as Cau II, Ben I cleaves the
Nci
different
position
generalization
their
the
that
(Roberts
1987).
There are
is
type II restriction enzymes cleave the
reminiscent
(Bickle
1987).
belong
to the
none
cleave
DNA
but,
at
to
DNA
of type I and
type
III
type II category.
restriction
First they require
Mg++
as
the
within
In some ways,
However, by two criteria 'enzymes such as Mbo
a
cut
the
enzymes
II
clearly
a
cofactor
of the other cofactor needed for type I and III and second, they
the
recognition
zymes
I,
excepti{)ns
DNA at some distance from their recognition sites.
but
Ben
recognition sequences. Several of these enzymes, such as Mbo II
behaviour
DNA
at
fixed
rather then
variable
sequence (Brown et al., 1980).
hydrolyse
droxyl groups.
phosphodiester
The
bonds
different aspects of
small number of
distances
All type II
away
from
restriction
to leave 5' phosphate and
3'
enhy-
type. II enzymes are thus a diverse and heterogeneous
group of proteins and individual enzymes
very
also
sequence
can be exploited to analyse
DNA-Protein interactions.
However, at present only a
restriction enzymes have been analysed in -any
chemical detail and in terms of
their recognition
to
X-ray crystal structure is
some extent EcoR V ; their
30
many
except for EcoR I
known
bioand
though
anything
har~ly
about the
cleavage mechanism is known.
(ii) Structure
Cocrystal structure of two restriction enzymes ECoR I and ECoR
with
their cognate sequence bound in absence of Mg++
have been
V
analysed
in great detail.
(a)
EcoR I : Of
best
the
characterized
symmetric
many
is the EcoR I system.
that
homodimer
The EcoR I
endonuclease
dimension
1989;
1984;
and
in
the DNA and enzyme may
occur
McClarin
·et
structures
studied
by
studies
al.,
of
1986; Kim
naked
these finding
(Lane
et al., 1991)
anomalies at EcoRI sites,
the
more
that
bends it to
substrate
flanking
DNA
et
the
a
structure
in
has
and
been
are
bound
or
EcoRI
50° angle (Kim et
a
unwinds
sites
1991).
with
analysis,
More
the
recent
structural
increased flexibility
conformation
EcoRI
backbone
flanking
al., 1984) .. In the revised
the original
the
structure and present
have disappeared (Frederick et al., 1984; McClarin et
al., 1990; Rosenberg 1991). Current
model
31
EcoRI
suggests
site,
in
the
25°
EcoRI-
al., 1986;
that
is
sites
DNA by approximately
central kink is present in
kinks observed in
al.,
EcoRI sites have been
containing
the phosphate
in
et
Rosenberg
controversial.
kinks
al.,
DNA-protein
elestrophoretic
consistent
one
Conformational
specific
1990;
oligonucleotides
kinks are present
while
a
recognition
1984; Frederick
al.,
protein
either showing
B-DNA. 3lp NMR of
naked EcoR I sites
and
in
flanking backbone. However overall DNA backbone
like
confirms
and
et
X-ray crystallography, NMR
interpretation of
in
Mclanghlin 1988).
to trigger DNA scission (Kim et. al.,
Although
NMR
scans
by recognizing distorted B-form DNA (Nerdal et
et al., 1991; Dickmann
complex
is
In vitro studies revealed EcoR
(Jacket al., 1982; Terry et al., 1985) to find its
Lane
changes
endonuclease
initially binds non-specifically and then
site GAATTC, possibly
the
cleaves the sequence GAATTC between G and A on
both DNA strands (Bennet and Halford 1989).
I
systems
known restriction-modification
but
naked
Kim
kinks
flanking EcoRI sites serve
Following
produce
features
of
and distorts DNA
flanking the
naked
the original structure, such as the DNA
site.
binding
However, other aspects differ; the amino
is widened by 3.5A 0 to permit access
the
increased
buckle,
site,
central
thymine
carboxy-
the major
Each
complex,
and an
groove,-
Several
the
proposed
al.,
1986;
al.,
1989;
and
GAATTC
adenines
the
and
and
anchors
DNA-
centre
the
the scissile bonds in the
chain that steeply penetrate and
of
DNA,
active
by
traverse
laboratories when tested the original model by
contact residues like glu144 arg145 and
et
arg200
al., 1989; Heitman and Model 1990;
Oelgeschlager
et al., 1990; Osuna et al.,
Conservative substitutions (arg200 __ lys,
substrate specificity, (Wolfes et al., 1986).
at
retained
considerable
DNA
mutations
and
respectively.
substitutions
hydrogen
rna-
residues that recognizes 'GAATTC' are borne
possible
in
major
the
monomer bears an arm that enwraps
extended
Needles
inconsistent.
Heitman
of
2 fold axis of symmetry through the
each subunit,
two ex-helices
alter
enzyme is a symmetric dimmer
a
sites.
the
In
I
shows
I
EcoR
sites.
DNA
angles
roll
the backbone is kinked at the centre
EcoR
complex
stabilizes
The
al.,
recognition
the
unusual
et
carbonyl oxygen alter the inner AT basepair conformations.
The
the
pairs have
Some
ex-helices,
and
the central hydrogen bonds between the central
and
enzyme
base
to
to
residues, and the
The DNA structure in the complex is quite unusual.
chinery,
ity
bends,
by EcoRI.
II neokinks flanking the EcoR I site have disappeared (Kim
groove
not
unwinds,
recognised
have moved, the arm is now composed of internal
1990).
the
initial signspot
central kink and undo kinks
are still present.
type
an
specific binding EcoR I
a
termini
as
-
glu144,
arg145 and arg200,
cleavage activity
and Model 1990). None were altered
vivo
or in vitro,
bond
the
affecting
even
in
though some
DNA site, in addition,
these three residues
32
altering
(Wolfes
et
Alves
et
1991)
found
glu144 __ gln)
Among 50
several
(Needles
et
it
did
to
60
mutants
al.,
1989;
their substrate. specificsubtitutions
while
severely
double
impaired
could
and
not
triple
catalysis,
they did
not alter
chlager
et
al., 1990;
The
roles,
rather
revised crystal structure (Fig. 8a,8b) reveals two
residues
to
glu144
is near DNA it is not bound to
the
different
adenines
but
and hydrogen bonds with other residues. Asn141
to bidentate hydrogen bond the adenines (GAATTC).
Arg200
was thought to hydrogen bond guanine, now cooperates with arg203
which
chelate
a water
molecule that binds guanine (Rosenberg 1991). This
is consistent with the finding
matic
activity
but
that
arg200
(Yanofsky
by
(Fig. 9).
Substituting
to
model
enzy1990).
lys (Heitman 1989) or gluta-
et al.,1987) inactivates EcoR I.
arg145 does bidentate hydrogen bond,
tions
substitution decreases
not substrate specificity (Heitman and Model
Arg 20 3 is essential because substitution
mate
to
contact DNA glul44 and arg200, fulfill
salt bridges
is now proposed
This lead
of the crystal structure.
proposed
forms
Oelges-
al., 1989;
revised
while
Osuna et al., 1991).
et
a
detailed analysis
originally
substrate recognition (Alves
As originally
proposed,
span, and recognize adenine N7 posi-
arg 145 by lysine or
cysteine
does
not
alter substrate specificity (Heitman and Model 1990), most probably because
contacts to adenine N6 amino groups
and thymidine suffice to
discriminate
substrate.
A
novel
DNA recognition element which has so
not
far
been
found in other DNA binding proteins, an extended chain composed of residues
137
to
142
contacts
amino
lies
within the DNA major groove and
makes
van
der
with pyrimidines and hydrogen bonds with pyrimidines and
groups.
Within this extended chain lies ala138.
adenine
alal38
main
chain keto oxygen qydrogen bonds with the cytosine N4 amino group
asn 141 hydrogen bonds to two adenines N6 amino groups while met 137
The
cytosine-5-position,
gly140
with
the outer thymidine
The
Waals
methyl
ala142
with
the inner thymidine methyl group interact by
van
forces
(Rosenberg 1991).· In addition to the pyrimidine contact
group
der
with
and
Waals
from
the
extended chain, van der Waals interactions are proposed between ilel97
and
the cytosine 5-position, and glnllS and the inner thymidine methyl group.
33
Figure , . Schematic backbone drawing of one subunit of (dimeric) £coR I
endonuclease and both strands of the .DNA in the complex. The arrows
represent~ str&nds, the coils represent o helices and the. ribbons re~resent
the DNA backbone. The helices in the foreground of the diagram are the
inner and outter recognition helices. They connect the third 8 strand to the
fourth and the fourth ~ stund to the fifth. The two helices also for~~~ the
centrAl interface with the other subunit. The amino·terminus of the
polyper-tide chain is in the arm near the DNA.
FIG-
9
(b) EcoR V :
its
Recently
cognate
shows
DNA
EcoR
(Winkler et al., 1991).
V to be a
structural
similarity
the
of
lack
Furthe rrno re,
tortions
DNA
amino
acid
appears
loop
catalysis
Therefore,
I-DNA
acidic
of
with
the
loops.
enzyme
However
no
is found as expected
~coRI
similarity
in unbound
would
In
bonds
may
between
both
from
protein.
since EcoR
and bound
I
form
the
distortions
complex
with
with bases
in
of
the
and EcoR
cognate
major
the
++
bindi,ng
scissile
bond.
V are
with DNA,
disquite
are
participate in Mg
the vicinity
be quite distinct between
(iii) catalytic Mechanism
Analysis
in
that
The DNA
complex.
residues which
are located
both
recognition
sequence
more like A-DNA.
it appears
unrelated
EcoRV and
specific hydrogen
makes
groove and three
and
between
from the EcoR
a
The structure
protein with protruding
~~~
elucidated
the structure of the DNA in EcoRV-DNA cocrystals shows
and
different
the structure of EcoR V has been
structurally
mechanism for DNA
the two enzymes.
!
of the mechanism of action of restriction enzymes
have
two types of DNA substrates either long DNA molecules of length
1-50
kilobase pairs or synthetic oligonucleotides usually between 8 and 12
base
used
pair
long
rial
plasmids, or phage or viral genomes fully sequenced, have
cessfully
(Greene et al., 1975).
used.
covalently
of
closed
cutting
very
at
been
a
copy
product formed by cutting one site remains a substrate
for
other
sites, and different sites on the same
kinetics
circle (ocDNA)
cut
the
(Halford
and
Johnson 1980).
DNA
The
can
show
reason
the
is cccDNA is that it reveals directly the mode
recognition
open
This simplifies the kinetic
suc-
been
circle of duplex DNA (ccc DNA ) that contains one
by the restriction
at the
bacte-
for
substrate
cleavage
of
analysis,
different
ideal
strand
the
range
For mechanistic studies the ideal substrate has
the recognition sequence.
otherwise
For large DNA a wide
enzymes.
of
DNA
The enzyme initially cuts just
one
site on the 'ccc' substrates to
generate
form. A subsequent reaction will then be required
second strand
of the recognition site, thus converting
34
the
the
to
oc
form
of the DNA
vert the
ccc
detected.
to the
Alternatively, the enzyme may
three forms
electrophoresis
of DNA can be separated from each
through agarose.
mixture
at
time intervals, and stopping
for cutting each strand can be
belled
DNA or densitometry of
this
very
turnover
and
transient
kinetic
amounts
type
to
parameters
jian
the
for
1987;
for
normal
The
for
and
these
of
for
enzymes.
enzyme,
Moreover,
and
these
the values of kcat and
act as a competitive
necessary
nature
of
cannot
be
compared
the
the
Halford 1982). Type II
pairs at
its
recognition site,
pairs.
In
addition,
on
DNA
DNA
can
restriction
kro values for their recognition sites
sequence specificity.
kro is that for Hha II;
between
recognition site
This
is
not
to
the
that
the
4
base
for the biological function but also is intrinsic
DNA
values
temperature
kro measured
the
state
However,
substantially·
DNA typically of the order of 1 nM.
highest
more base
the reaction at
and
generally display low
plasmids or phage
only
to
et al.,l980; Maxwell
endonuclease
on
inhibitor
with
Chirik-
1976).
macromolecule will always be apparent values, as the rest of
(Langowski
and
For each enzyme
differ
turnover
steady
are determined under reaction condition and
that
time.
format
the
enzyme
1983;
cataly-
catalytic
Modrich and Zabel
with those for the other enzymes.
suitable
single
measures
of enzyme
Michaelis-Menten
reasons the constants for each
~
almost
given
several of these enzymes are known (Nardone
directly
kcat
As
both
at a
DNA concentration, and value for
Imber and Bickle 1981;
various
to
behaviour
kinetics
II enzymes follow the standard
respect
time
radiola-
This assay simply
of ccc, oc, and linear forms of the DNA
(Modrich and Roberts 1982).
by
the
experiments (Halford and Johnson,
Type II restriction enzymes show the
sis
either
by
number (Kcat < 10 min-I
minor modification
Terry et al., 1985; Halford and Goodell 1988).
the
other
reaction,
stained gels.
low turnover
method can be adapted with
the
obtained by using
ethediurn bromide
all restriction enzymes have a
being
Hence, by removing aliquots from
course
),
con-
substrate directly to linear DNA without any oc forms
The
reaction
linear form.
It may
this enzyme
where
as all
be
significant
must interact with
others interact with 5 or
restriction enzymes
35
have
much
lower
Kcat
values,
values
typically
1 min-I than the
majority
of
where
enzymes
of above 100 sec-1 are commonplace (Fersht 1985).
Given the similarities in the steady state parameters for different restriction enzymes, it might be anticipated that all of these
use
the same mechanism to cleave the DNA at their
sites,
this is not so (Halford 1983).
At least two
recognition
basic
mechanism
been described for different restriction enzymes:either the
have
of
but
respective
enzymes
each
cleavage
strand of the DNA take place by individual reaction that
separated
kinetically,
or
the cleavage of both strands
by
a
can
be
concerted
reaction in which the cutting of one strand cannot be separated kinetically
from that of the other strand. However, the categorization of a restriction
enzyme to one mechanism or another is essentially arbitrary.
A restriction
enzyme can cleave one DNA substrate by one mechanism and another
substrate
by
different
the
other, or alternatively it may cleave one substrate
mechanisms
under
different
by
reaction conditions ( Maxwell and
Halford
1982; Halford and Goodall 1988).
The first use of- a synthetic oligonucleotide as a
substrate
for
This
study
three principal characteristics of these
substrates.
These
the synthesis of a self-complementary
oligonucleotide
that
contains the recognition sequence, so that it can anneal to itself to
form
for
the
and this substrate gave a non-linear Lineweaver-Burk
plot
a
demonstrated
were,
a
enzyme
restriction
first,
duplex.
Reduced
oligonucleotide
was
by Greene
activity
was
et al.,
observed
(1975).
above
the
Tm
until corrected for the fraction of single-stranded material.
value
enzyme
of
Kcat
was
for cleavage of this oligonucleotide
same
as that for the
DNA
by
macromolecule.
the
Second,
restriction
However,
another
oligonucleotide substrate for the same enzyme with different flanking
p~irs,
gave
a
much
higher
value
of
Kcat
the
(Brennan et al.,
base
1986).
Oligonucleotides may well be cleaved by restriction enzymes faster than DNA
macromolecules,
steps:
an
for, these enzymes may dissociate from the latter
initial
transfer of the enzyme from the
36
recognition
in
two
site
to
nonspecific DNA, which then retains the enzyme until the final dissociation
(Terry
et al., 1985). Obviously facilitated diffusion can play no part
in
the reaction with oligonucleotides.
(C) LITERATURE REVIEW ON Bam HI:
The type II restriction endonuclease Bam HI from Bacillus
liquefaciens
3'
recognises
(Wilson
and
subject
of the
pressed,.
its
Many
the
symmetrical
sequence
Young 1975; Roberts et al., 1977).
pr~sent
X-ray
biochemical
duplex
5'-GGATCC-
This
enzyme,
thesis, has been cloned,sequenced
crystal structure has still not
and kinetic
data
are,
however
amylo-
and
been
the
overex-
elucidated.
available as discussed
below.
The
restriction
and
specific
endonuclease
modification system in bacillus
I
is
a
part
of
amyloliquifaciens
by
hydrolysis
of the
phosphodiester bonds
fashion
across
the
hexanucleotide
termini
(Wilson
and
Young
a
cofactor (Roberts et
sequence
1975).
al.,
in
generating
the
H (Wilson
Young 1976; Shibata and Audo 1976; Shibata et al.,l976). DNA
stricted
as
BamH
a
is
staggered
5'-phosphoryl
For catalysisy Bam HI requires
1977).
The mechanism
re-
of
cleavage
Hg++
can
be
explained as follows
(E+E).(S+S)
-->
(E+E).(S+P)
-->
(E+E)
(P+P).
Here E+E is the dimeric enzyme and S+S is the duplex DNA that is cut in one
or
both
the
transient
both
strands to yield S+P and P+P, respectively.
existence
means that the dissociation of the product
cut
strands (P+P) must be the slowest step in the pathway, and this
determine
the
transient
phase
the
phase
The
steady-state
means
rate. The absence of oc DNA
(S+P)
from
that the formation of this intermediate
first strand) is much slower than its breakdown (cutting
. 37
of
in
must
the
(cutting
the
second
-
strand).
Gel
filtration of Bam HI endonuclease at ionic strengths
0.3 M NaCl and
lar weight
protein concentration of O.lmg/ml shows
of 90,000.
was estimated
ugation.
a
native
In the presence of 0.5 m NaCl the molecular
SDS-PAGE
revealed
a single band with
weight
centrif-
weight
molecular
of
These suggests that the endonuclease consists of identical
polypeptide chains which aggregate to dimerise or tetramerise depending
strength.
ionic
th~
to
molecu-
to be 46,000 by gel filtration and sucrose density
22,000 daltons.
up
range,
with
1OrnM.
The addition
Bam HI endonuclease exhibit activity over
optimuin at 8.5 in tris buffers.
an
The
on
broad
pH
Mg++
is
optimum
Tween 20 or BSA to the reaction solution
greatly
activity at 37°C. The enzyme is most active at
37-40°C
and
appears to be stable to thermal denaturation upto 45°C in the
absence
of
substrate
in
stabilizes
presence
enzyme
of
or
of
Nacl.
However, the enzyme is
lOOml Nacl.
salt 10-50 mM
lower
It can be further stablized
to
65°C
optimally
the
active
and is severely inhibited at 250 mM Nacl.
DNA
at
also
has a stabilizing effect at 37°C when protein solutions are dilute (Nardone
and Chirikjian 1987).
The _purified
buffer
Mg ++ at
Mn++
zn++
with loss
cu++ could
performed
with form
site)
substitute for Mg++.
not
estimated turnover
shown
to
al.,
data
subsets
be
containing
GG
studies
The 5'-phosphoryl
(Lee and Chirikjian
HI
nm.
deoxydi-
Bam HI sequence (GG,GA,AT,TC,CC) have
inhibitors
et
1979;
been
George
al., 1980) reciprocal plots of representative
and CC indicated
unrelated
·could not inhibit
varied
the
specific
Hinsch
Initial velocity
with Kms in the vicinity of 3.6
number was 1.5 min-I.
of
1985;
Dinucleotides
kinetics
re-
replaced
Sv40 or pBR-322 DNA (both containing one Bam
I
in
Other divalent cations like ca+ ~
of 80% of activity.
revealed hyperbolic
nucleotide
et
year
optimal concentration of lOmM which may be
an
and
The
one
containing 50% glycerol (Smith and Chirikjian 1979).The enzyme
quires
by
enzyme is quite stable for at least
to the Bam
the endonuclease.
over wide range suggesting
competitive
HI sequence
The
inhibition
such as AA,TT,
Ki values
patterns.
and
of the dinucleotides
differences in the interactions of
38
GC
the
endonuclease
at
various points within the
dinucleotides CC and GG
had the
ends
site are
of the recognition
The inhibiting effects
of
most
recognition
potent
most
Kis, suggesting
important
the dinucleotides were found
changes in the enzyme caused
the binding of another.
for
the
the
inhibitors. The
the
symmetrical
to
conformational
meas-
SV40 DNA complex (formed
>1 suggesting multiple
construct Hill
interacting
existence of at least two binding
active site for each inhibitor
to
to be synergistic
the dinucleotide were used to
Hill coefficients were
activity.
filter binding experiments
stable endonuclease
by
Mg++)
the
binding of an inhibitor which affects
Nitrocellulose
uring the displacement of
plots.
by the
The
that
for enzyme
in mixing experiments. This has been presumed to-be due
in the absence of
sequence.
sites
sites
in
has been taken into consideration
due
double stranded nature of BamH I
site
(Nardone
and
Chirkijian 1987).
The kinetics o(, Bam HI endonuclease was examined with full length
pBR322
DNA that had been linearized with different
restriction
enzymes.
The endonuclease exhibited faster reaction rates with substrates having the
recognition
site
Experiments
that
with
or 5')
of
position
(Nardone
et
Km
the
If
or
target
the nearest end), it is
This
would be
for
ascertain
Bam HI,
enzymes
direction
DNA
a
a
and
1921
presumed
that
more types of facilitated
can locate their recognition
site
by
then an enhancement of these process
manifestation
non-specific
binding
if facilitated
centrally located
the
Bam HI
a
would
central
of the proportionately
around
a
compared
occur as the recognition site is located in a more
area
to
these
hoping mechanism
be expected to
position.
from
showed
There was
(Bam HI Cleavage site is 375
rate differences originate from one or
diffusion.
sliding
for EcoRI linearized pBR322
pBR 322 DNA.
pairs, respectively,
reaction
order
in
Ndei linearized
base
al.1986).
a variety of linearized pBR 322 DNA substrates
the nearest DNA terminus to the Bam HI site.
fold increase
to
the
a more central
these effects were connected with the distance and not the
(3'
3
in
longer
site.
In
diffusion is kinetically evident
for
Bam HI site on linearized pBR 322
was
39
used. It was
74
observed
bps to 4362
bps
that when
the
cleavage rates
increases
folds.
cleavage
In
absence of NaCl the kinetic preference for longer
observed
that
greatly reduced
by 9
between
the
rates
substrate length is increased
Differences
in the presence of 160 mM
was rate limiting the increases in
Nacl.
substrates
over a DNA concentration range of O.l-12nm. With
association
from
the
the
was
assumption
cleavage
rates
could have been due to the longer target area for non-specific binding and,
therefore, an increase in the frequency of facilitated transfer. The elimination
rates
of
both
between
consistent
cific
differences
the full length substrates by high
with a decrease in the
cleavage
the
I
activity.
of
of
non
second,
Ba~
This secondary activity called
activity of the same enzyme but is induced by
Bam
HI
HI.l
has been seen to be altered in
the
presence
of
hydrophobic
from the Bam HI palindrome.
High alkaline conditions
the enzyme
uifaciens
(George et al., 1980).
strains
strains
1976
F
apart
However,
various
from strain H have
), while
strain
N
showed
two
activities
called Bam NI and Bam Nx (Shibata,
having
same
the
molecular
discrete
The
Mg++
weight
bands
specificity as Bam HI,
than
Bam
as compared to
concentration
Nx
the
and
was
distinct
to
cleaved
phage
latter which
gave
requirements
for optimum
40
to
Bam
to
amongst
(Shibata
et
endonuclease
and Audo 1976 ).
found
served
B.amyloliq-
found
and · K showed same cleavage specificity
in-
inherent
other
.been
were
and
But the conversion of Bam HI
is found to be reversible and therefore the property is
which
DNA
enzyme concentration in the presence of organic solvents
optimize the secondary activity.
the
The specificity of cleavage
different
creased
of
to
or the activity
such as glycerol and DMSO. Cleavage sites in 0 174 RF
al.,
spe-
was found
reagents,
HI.1
is
distinct
alteration
It is also termed as "Star activity"
observed due to relaxed specificity of Bam HI.
to
cleavage
concentration
electrostatic component
seems to have a trace- amount
reaction conditions.
of
NaCl
in
binding.
BamH
be
long chain preference and the
have
A
BamN
a
lower
into
five
fifteen
activity
I
were
bands.
found
to
different, also, phage 105-C.H
be
by
protected
showing
Barn NI
of
while Ban Nx gave eight bands instead
fifteen
the degeneracy of the reaction.
Amino
various
acid residues required for activity have been
biochemical
sensitivity
and
completely
grown on strain H was
methods. Bam HI endonuclease was
studied
found
to
by
have
towards reagents that modify sulphydryl groups. At neutral
pH
37°C, incubation of the enzyme with 6mM ioodoacetamide, dithiobis
(2-
nitro benzoic acid) or N-ethylmalemide for 45 minutes resulted in 45%,
23%
and 30% inhibition of activity respectively.
Using 5,5'-bis (2-nitobenzoic
acid)
DTNB and p-mercuribenzoate it has been shown that Bam HI
lost
its activity (Wells, et al., 1981).
endonuclease
that
contain
the
nucleotide or phosphate-binding sites (George
an
which were
nition
arginine specific reagent, inhibited
presence
preliminary
of
sequence.
inactivation.
recognition
These studies
enzymes
role of these amino
al.,1985)
by
inhibitors
HI palindrome.
acids in
enzyme
most
Dinucleotides that were
failed to protect the enzyme
~hat
arginine residues may
the
from
reside
sequence-specific
Different variants of Bam HI
that
cleavage activities were found to
have
cl~ave
catalysis. The active site has been shown to
charged pocket comprising largely
of
Aspartate
coordination complex with divalent Mg++ ions
the process of catalysis.
pyridoxal
and
during
The loss of these moieties allows the enzyme to
(Shuang Yang
lysyl residue also can
with
indicate
the
and aspartate residues indicating direct or indirect
negatively
bind but not to
sequence
undetectable
Glutamate which forms
residues
proteins
decreased
competitive
active site and might function in
mutation at glutamate
of
HI
endonuclease
was
dinucleotide pdGpdG protected
the butanedione modification.
displayed reduced or
a
et
the
The inhibition
of the enzyme with DNA or
The
recognition of the Bam
have
Bam
the 5'-phosphoryl deoxydinucleotide subset of the Bam HI recog-
unrelated to the
the
sodium borate.
incubation
efficiently against
in
in
have been examined because of their alleged role in
Butanedione,
in
Arginyl residues
completely
not
Xu
be ruled
and Ira Schildkraut 1991).
Role
out since modification of
lysyl
phosphate inhibit Bam HI
and Chirikjian 1987).
41
activity
(Nardone