Content uploaded by R. Stephen Lloyd
Author content
All content in this area was uploaded by R. Stephen Lloyd
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
0
1989 by The American Society
for
Biochemistry and Molecular Biology, Inc
THE
JOURNAL
OF
BIOLOGICAL CHEMISTRY
Val.
264,
No.
29, Issue
of
October
15,
pp.
17422-17427,1989
Printed
in
U.
S.
A.
Modulation
of
the
DNA
Scanning Activity
of
the
Micrococcus
luteus
UV
Endonuclease*
(Received for publication, May 8, 1989)
School of Medicine, Nashuille, Tennessee 37232
”
Micrococcus luteus
UV endonuclease incises DNA at
the sites of ultraviolet (UV) light-induced pyrimidine
dimers. The mechanism of incision has been previously
shown to be a glycosylic bond cleavage at the 5“pyrim-
idine
of
the dimer followed by an apyrimidinic endo-
nuclease activity which cleaves the phosphodiester
backbone between the pyrimidines. The process by
which
M. luteus
UV endonuclease locates pyrimidine
dimers within a population of UV-irradiated plasmids
was shown to occur,
in vitro,
by a processive or “slid-
ing” mechanism on non-target DNA as opposed to a
distributive or “random hit” mechanism. Form
I
plas-
mid DNA containing 25 dimers per molecule was in-
cubated with
M.
luteus
UV endonuclease in time course
reactions. The three topological forms of plasmid DNA
generated were analyzed by agarose gel electrophore-
sis.
When the enzyme encounters
a
pyrimidine dimer,
it
is
significantly more likely to make only the glyco-
sylase cleavage
as
opposed to making both the glyco-
sylic and phosphodiester bond cleavages. Thus, plas-
mids are accumulated with many alkaline-labile sites
relative to single-stranded breaks. In addition, reac-
tions were performed at both pH
8.0
and pH
6.0,
in the
absence of NaC1, as well as 25,100, and 250 mM NaC1.
The efficiency of the DNA scanning reaction was
shown to be dependent on both the ionic strength and
pH of the reaction. At low ionic strengths, the reaction
was
shown to proceed by
a
processive mechanism and
shifted to
a
distributive mechanism
as
the ionic
strength of the reaction increased. Processivity
at
pH
8.0
is
shown to be more sensitive to increases in ionic
strength than reactions performed at pH
6.0.
The process by which DNA binding proteins locate and
recognize their target sequences within large domains of DNA
has been of considerable interest in recent years. Several
proteins have been shown to locate their target recognition
sites by using a facilitated diffusion mechanism in which the
protein is able to scan
or
slide along DNA in
a
processive
manner (for reviews, see Refs. 1-3). Examples of these types
of proteins are, lac repressor (4-9), T4 endonuclease V (10-
12),
EcoRI
(13-15), BarnHI, BamHI methylase (16), and RNA
*This research was supported in part by United States Public
Health Service Grants ES 04091 and ES 00267. The costs of publi-
cation of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “aduertise-
ment” in accordance with
18
U.S.C. Section 1734 solely to indicate
this fact.
7
Predoctoral fellow supported by the Molecular Biology University
Scholarship and the Harold Sterling Vanderbilt Award.
**
To whom correspondence should be addressed. Tel.: 615-322-
3063.
Richard
W.
HamiltonSgll and
R.
Stephen Lloyd§ll**
From the Departments
of
$Molecular Biolocv and
II
Biochemistry and the §Center in
Molecular
Toxicology, Vanderbilt University
polymerase (17-22). An additional facilitated diffusion mech-
anism which has been proposed is a looping model (for re-
views, see Refs.
1
and 23). This model allows proteins to
transfer between separate segments of DNA. Facilitated dif-
fusion mechanisms allow proteins to locate their recognition
sequences at a rate as much as 1000-fold faster than would be
expected for simple three-dimensional diffusion (3). These
mechanisms involve electrostatic interactions between the
protein and the DNA helix and are sensitive to changes in
ionic strength (2). As ionic strength increases, the dissociation
rate of the protein with nonspecific DNA sequences also
increases. Therefore, low ionic strength conditions increase
the amount of time the protein is bound to nonspecific DNA
sequences, and, as a result, the protein is able to diffuse larger
distances along the DNA.
One of these DNA-reactive enzymes, bacteriophage T4
endonuclease V, is a pyrimidine dimer-specific endonuclease
which has been shown to scan DNA in a processive manner
both
in vitro
and
in vivo
(10-12,24,25). The
in
vitro
scanning
reaction has been shown to be sensitive to increases in ionic
strength with processivity being abolished at salt concentra-
tions higher than 50 mM NaCl at pH 8.0. In addition, site-
directed mutagenesis has been performed on portions of the
denV
gene, which encodes endonuclease V. Analyses of some
of the mutant enzymes has shown that the alteration of
certain positively charged amino acids abolishes the ability of
the enzyme to act processively on non-target DNA
in vitro
(25). Studies of these mutated enzymes
in vivo
have shown
that, unlike wild type endonuclease V, they are unable to
confer enhanced UV resistance to UV-sensitive
Escherichia
coli
hosts. Thus, the ability to scan non-target DNA
in vivo
is of biological importance.
Micrococcus luteus,
a
Gram-positive cocci, produces an en-
zyme, UV endonuclease, which has several similarities to T4
endonuclease V. Both proteins are small molecular weight
proteins (18,000 and 16,000, respectively), and neither shows
any requirement for divalent cations
or
ATP. These enzymes
have been shown to incise UV-irradiated DNA at the site of
pyrimidine dimers by cleaving the 5’-glycosylic bond followed
by a type
I
apyrimidinic endonuclease activity which cleaves
the phosphodiester backbone between the two pyrimidines of
the dimer (26-33). This two-step mechanism is unique to
these enzymes. Due to the many similarities exhibited by
these two enzymes, we have investigated the
in vitro
DNA
scanning ability of the
M. luteus
UV endonuclease and how
this activity is affected by changes in pH as well as ionic
strength.
MATERIALS
AND
METHODS
Purification
of
M. luteus
UV
Endonuclease-Freeze-dried M. luteus
cells (ATCC 4689) were obtained from Sigma and resuspended in 500
ml of 10 mM Tris-HC1 (pH 7.5), 10 mM EDTA, and 25 mM
KCI.
Cells
17422
DNA Scanning Activity
of
M.
luteus
UV
Endonuclease
17423
were treated with lysozyme and sonicated briefly to shear the large
genomic
DNA.
Cellular debris was removed by centrifugation. The
UV
endonuclease was purified
to
homogeneity by sequentially using
the following chromatographic steps: single-stranded DNA-agarose,
Sephacryl
HR200,
and fast protein liquid chromatography Mono
S.
Details of this purification scheme are
to
be published elsewhere.
Kinetic Analysis
of
M.
luteus
UV
Endonuclease with UV-irradiated
DNA-Form
I
[3H]pBR32Z in 20 mM EDTA
(pH
8.0)
at
1
pg/d
was
irradiated by
short
wave (254
nm)
UV
light at
100
microwatts/cm*
for 245
s
in order
to
generate 25 pyrimidine dimers per plasmid
molecule. This DNA was then added
to
various buffers (described
below), at
a
final concentration of
0.1
pg/pl.
All
kinetic analyses were
performed at substrate concentrations below
the
K,,
as revealed hy
altering
the
reaction velocity by altering the number
of
pyrimidine
dimers per
molecule
(data not shown).
All
buffers contained 5%
glycerol,
0.1
mg/ml
BSA,’ and various concentrations of
NaC1.
The
pH
of
these buffers was modulated with one
of
the following:
10
mM
Tris-HC1 (pH 7.5),
20
mM
Tricine (pH
8.0),
or
20
mM
MES (pH
6.0).
M.
luteus
UV
endonuclease was added at appropriate concentrations
to
allow the reactions
to
be monitored
over
a
30-min kinetic analysis.
At the indicated time points,
1O-pl
aliquots of the reaction mixture
were removed and added to the appropriate stop buffer. The stop
buffers used contained
50%
sucrose, 2% SDS, 50
mM
Tris-HCl (pH
8),
20 mM EDTA, and bromphenol blue. Alkaline hydrolysis
was
achieved by adding
1
N
NaOH to this stop buffer
to
a final concen-
tration
of
1%.
The final pH
of
the reaction is approximately pH
11.
This pH results in the cleavage of the phosphodiester bond
at
apu-
rinic/apyrimidinic (AP) sites without denaturing the DNA. For the
analyses which were performed
using
denaturing gels, the
stop
buffer
contained 40% sucrose,
1%
SDS,
1
mM
EDTA, and
100
mM NaOH.
These conditions not only
cleave
the phosphodiester bond at
AP
sites,
but also denature the DNA. The methoxyamine stop buffer contained
200
mM
methoxyamine,
0.5%
SDS,
and 30% glycerol. Methoxyamine
treatment renders AP sites impervious to alkaline hydrolysis
(34),
and
thus
the
complete
incision activity of
the
enzyme
can
be moni-
tored utilizing denaturing agarose gels without
the
concomitant
hy-
drolysis
of
alkaline-labile sites.
In order
to
investigate the pH optimum for
the
enzyme, reactions
were performed in the following buffers at the indicated pH values:
Na+-acetate (4.5, 5.0), MES (5.0, 5.5,
6.0),
PIPES (6.0,
6.5),
MOPS
(6.5,
7.0),
HEPES
(7.0,
7.5),
Tricine
(7.5,
8.0,
8.5), CHES (8.5,
9.0),
and CAPS
(9.0).
A
wide range
of
buffers with overlapping pH values
were used
to
eliminate
the
possibility
of
buffer-specific effects. The
final concentration
for
all these buffers was 50
mM,
and the ionic
strength of the reactions was
100
mM
NaC1. Single time points were
taken after 20 min and terminated with and without the alkaline
nondenaturing treatment. These reaction products were
then
ana-
lyzed by agarose
gel
electrophoresis.
All
reaction products were resolved
by
electrophoresis in 1.2%
agarose gels. The
gels
were stained in
1
pg/ml
ethidium bromide in
electrophoresis buffer consisting
of 40
mM sodium
acetate
and
2
mM
EDTA. For the native and nondenaturing alkaline treatmehts,
the
DNA hands were visualized by exposure
to
long wave
UV
light. Form
I,
11,
and
III
DNA bands were excised from the gels and counted
by
liquid scintillation spectroscopy
(10).
In order
to
determine
the
DNA
fragment size distribution which was generated by t.he enzyme,
sam-
ples
were resolved by electrophoresis in denaturing agarose gels.
These gels were presoaked in 30
mM
NaOH and
1
mM
EDTA
overnight to
ensure complete
denaturation
of
the
DNA as well
as
alkaline hydrolysis
of
any remaining AP sites. Following the separa-
tion
of
single-stranded DNA fragments by electrophoresis, the dena-
turing
gels
were processed by the method
of
Southern (35, 36).
RESULTS
Investigation
of
the DNA Scanning Ability
of
M.
luteus
UV
Endonuclease-In order to investigate the in vitro DNA scan-
ning ability of the UV endonuclease, kinetic analysis of the
enzyme with UV-irradiation plasmid DNA was performed as
The abbreviations used are: BSA, bovine serum albumin; AP,
apurinic/apyrimidinic;
SDS,
sodium dodecyl sulfate; MES, 2-(N-
morpho1ino)ethanesulfonic
acid; PIPES,
piperazine-N,N-bis(2-eth-
anesulfonic acid),
MOPS,
3-(N-m0rpholino)propanesulfonic
acid;
HEPES,
N-2-hydroxyethylpiperazine-N”2-ethanesulfonic
acid;
CHES,
2-(cyclohexylamino)-ethanesulfonic
acid; CAPS, 3-(cyclohex-
y1amino)propanesulfonic acid.
described under “Materials and Methods.” For a reaction
mechanism in which an enzyme may function by
a
processive
nicking activity, plasmids should accumulate which have been
completely incised at all the dimer sites. Thus, one would
expect to see a significant amount of form
111
DNA generated
while there is still
a
large percentage of form
I
DNA remain-
ing. In a distributive
or
random hit mechanism, almost all the
form
I
DNA
should be converted to form
I1
DNA before
significant amounts of form
111
DNA are generated.
Fig.
1
shows the results of these analyses performed at salt
concentrations of
0,
25,
100,
and
250
mM NaC1. The rate of
loss of form I DNA is most rapid for the
25
and
100
mM NaCl
reactions and slowest
for
the
0
mM reaction, while the
250
mM reaction is intermediate. Under these conditions, no form
I11
was observed to accumulate for any of the salt concentra-
tions tested, a result which was contrary to what would be
expected if the enzyme was acting in a processive manner.
These reactions were also analyzed by determining the
distribution
of
single-stranded DNA reaction products on
denaturing agarose gels as described under “Materials and
Methods.” By utilizing denaturing gels, the size distribution
of
the DNA fragments generated by the enzyme can be
visualized. The size of the DNA fragments which were gen-
erated corresponds to the distance between incised dimer
sites. If small fragments were produced while significant
amounts of form
I
DNA still remain, this would indicate that
adjacent dimer sites have been cleaved and that the enzyme
was acting in a processive manner. Also, the alkaline condi-
tions which are present in the denaturing gels will cleave any
AP sites which were generated by the glycosylase activity and
not completely incised by the enzyme. The results of this type
of analysis are shown in Fig.
2.
Panel A shows the loss of
form
I
and form
I1
DNA
for time course reactions performed
at
25
mM and
100
mM NaC1. The DNA fragment size distri-
bution which was generated is not evident in panel
A
but
is
present in
a
longer exposure (panels
B
and
C).
The major
difference between the two reactions shown in panel A is that
for the
25
mM NaCl reaction the form
I
and form
I1
DNA are
lost at approximately the same rate throughout the time
course, whereas in the
100
mM NaCl reaction, the form
I
DNA is rapidly converted to form
I1
DNA, followed by a slow
loss in form
I1
DNA. Panel
B
shows the distribution of
fragment size generated for both the
25
and the
100
mM NaCl
reactions
at
the same
(20
min) time point. This shows that
for the
25
mM reaction (lane
B),
much smaller fragments are
being generated than those observed
for
the
100
mM reaction
(lane
C).
In addition, it should be pointed out that there is
still form
I
DNA remaining in the
25
mM reaction, while in
the
100
mM reaction all the form
I
DNA
has been incised to
generate form
I1
DNA. Panel
C
shows the distribution of
fragments generated for the
25
mM and
100
mM NaCl reac-
tions
at
the point where equal amounts
of
form
I
DNA are
remaining for both reactions. This type of comparison allows
the examination of how many single-stranded breaks (AP
endonuclease and alkaline hydrolysis) are generated per en-
zyme-DNA encounter. As seen in panel
C,
the
25
mM NaCl
reaction generated
a
distribution
of
smaller fragments relative
to that observed at the
100
mM NaCl reaction. This suggests
that for each enzyme-DNA encounter in the
25
mM NaCl
reaction, the enzyme is acting
at
the sites of several if not all
the dimers present in that molecule. By comparison, the
100
mM NaCl reaction has generated only form
I1
DNA suggesting
that for each enzyme DNA encounter, it is generating only
one
or
a very few single-stranded beaks.
Modulation
of
DNA
Scanning in Vitro by Ionic Strength-
Due to the apparent discrepancy between the fragment size
17424
DNA
Scanning Activity
of
M.
luteus
UV
Endonuclease
liK
lOOmM
NaCl
40
20
0
0
10
20
30
1
time (min)
OmM NaCl
il;-El
:E
m
FIG.
1.
Kinetic analysis
of
M.
lu-
teus
UV Endonuclease on UV-irra-
diated DNA.
Form
I
[:'H]pRR322 in 20
a
0
40
mM EDTA at
1
pg/pl was irradiated by
short wave (254 nm)
UV
light at
100
E
20
microwatts/cm'
for
245
s
in order to
e
generate 25 pyrimidine dimers per mol-
$0
ecule. This DNA was incubated with pu-
time tmin)
rified
M.
luteus
UV
endonuclease in time
E
2!
a
z
z
0
C
al
.-
c
e
0
10
20
30
40
P
course reactions. The three topological
forms of DNA were separated by agarose
gel electrophoresis and quantitated by
liquid scintillation counting. Reactions
were performed in
10
mM Tris-HCI (pH
T.5),
S%
glycerol,
0.1
mg/ml BSA, and
the indicated concentration of NaCI.
Symbols
used are: form
I.
0:
form
11,
0,
form
111,
m.
250mM
NaCl
rn
25mM
NaCl
40
20
C
e
$0
0
10 20
30
40
time (min)
a
z
0
0
10
20
30
40
time (min)
Panel
A
Panel
B
Panel
C
AB
25mM
time
(min)
0
5
10
15
20
30
lOOmM
time
(min)
0
5
10 15 20
30
ABC
-
form
I1
form
I
form
I1
form
I
form
II
m
Y
form
I
form
II
4
form
I
FIG.
2.
Analysis
of
the distribution
of
single-stranded breaks produced
by
M.
luteus
UV endonuclease
within a population
of
UV-irradiated DNA.
Panel
A,
kinetic analysis reactions identical with those shown in
Fig.
1
were performed, and the reaction products were separated on denaturing agarose gels. Reactions were
performed in
10
mM Tris-HCI (pH i.5), 5% glycerol,
0.1
mg/ml BSA, and the indicated NaCl concentration.
Panel
H,
identical time points from
panel
A
were selected for comparison of the 25 mM and
100
mM NaCl reactions.
Lane
A,
0
min.
Lane
B,
25 mM, 20 min.
Lane
c,
100
mM, 20 min.
Panel
C,
time points with the same amount of form
I
DNA remaining for both the 25 mM and
100
mM NaCl reactions
(panel
A)
were selected
for
side-by-side comparison.
Lane
A,
25 mM NaCI.
Lane
H,
100
mM NaC1.
generated in the denaturing gels (Fig.
2)
and the lack of form
I11
generated in the native agarose gels (Fig.
I),
it was hy-
pothesized that the AP endonuclease activity was much less
active than the glycosylase activity under these conditions
(Tris-HC1, pH
7.5).
A difference in the activity levels has
been reported previously
(37).
If this were the case, many
apyrimidinic sites would be created by the enzyme because
the phosphodiester bond between the pyrimidines would not
be incised to generate
a
complete break. In order to test this
hypothesis, we performed time course reactions identical with
those described in Fig.
1.
These reactions were terminated
with SDS and then treated with alkali to hydrolyze the
phophodiester bond at any remaining AP sites. Fig.
3
shows
a
kinetic analysis of the reaction products obtained under
these conditions. All the salt concentrations tested showed an
increase in the amount of form
I11
DNA that was generated.
However, the
0
and
25
mM NaCl reactions show
a
significant
accumulation of form
I11
DNA while there is still form
I
DNA
remaining; by contrast, the
100
and
250
mM reactions do not
start to accumulate significant amounts of form
I11
DNA until
nearly
all
the form
I
DNA has been converted to form
11.
This
is highly consistent with what would be expected for a pro-
DNA Scanning Activity
of
M.
luteus
UV
Endonuclease
17425
FIG.
3.
Kinetic analysis
of
M.
lu-
teus
UV
endonuclease using alka-
line hydrolysis.
Reactions identical
with those described for Fig.
1
were per-
OmM
NaCl
1i;bl
z
0
40
g
20
z
$0
-
0
10
20 30
40
time (min)
lOOmM
NaCl
z
n
40
g
20
n
0
10
20
30
40
time (min)
formed. After terminating the reactions
with
SDS,
alkaline hydrolysis of the re-
25mM
NaCl
250mM
NaCl
maining apyrimidinic sites was achieved
by adding NaOH to pH
11.
Symbols are;
form
I,
0;
form
11,.
form
111,
..
~1:f"-g
~
1f-fy"y
4
60
40
-
c
5
20
c
a
no
K
n
0
10
20
30
40
time (min)
0
10
20
30
40
time (min)
cessive reaction mechanism occurring at the lower
(0
and
25
mM) salt concentrations and a more distributive mechanism
occurring for the higher
(100
and
250
mM) salt concentrations.
Since most incisions at dimer sites are the glycosylic bond
scission rather than the combined glycosylase/AP incision,
the processive reaction refers only to the scanning of the
enzyme on non-target DNA which facilitates dimer binding
and glycosylic bond scission. This is in contrast to the T4
endonuclease V enzyme in which the complete nicking reac-
tion is processive.
Quantitation
of
Glycosylase Versus AP Endonuclease Actiu-
ity-In order to quantitate the difference between the activi-
ties of the glycosylase and the AP endonuclease, the -In of
the rate of form I loss for both the native and alkaline-treated
reactions were compared. Under distributive conditions
(100
mM NaCl), the -In of the rate of form I loss should equal the
average number of single-stranded breaks generated. By com-
parison, we were able to calculate that under these conditions
the glycosylase is approximately 3 to
5
times more active than
the AP endonuclease. This ratio appears to be constant over
the salt concentrations which were evaluated in these exper-
iments (data not shown). It should be noted that this value
varied over time and with different enzyme preparations
suggesting that the AP endonucleolytic activity is more labile
over time than is the glycosylase activity.
Quantitation
of
pH
Optimum in Vitro-In order to investi-
gate the possibility that the glycosylase and the associated
AP endonuclease might have different pH optimum in uitro,
reactions similar to those previously described for the UV
endonuclease were performed across a wide pH range utilizing
several different buffer systems. These results are shown in
Fig. 4. Panel
A
shows the percentage of form I DNA remaining
after
20
min for both native and alkaline-treated reactions.
All reactions were performed under conditions which result
in a random incision of dimers at pH
8.0
(100
mM NaCl).
There is an apparent pH optimum of between
6
and
8
for the
glycosylase activity, with a slightly larger loss of form I DNA
observed for the alkaline-treated samples. More striking are
the differences in the amount of form I11 DNA generated
during these reactions as shown in Panel
B.
Modulation
of
DNA
Scanning in Vitro
by
Changes in
pH-
The large amount of form I11 DNA generated at pH
6
led us
to postulate that the enzyme may be acting processively at
this lower pH in spite of the high
(100
mM NaC1) salt
conditions. In order to investigate this possibility, kinetic
analyses, identical with those previously described, were con-
ducted with the exception that the reactions were performed
at both pH
6
and pH
8
for the same salt concentrations
(0,
25,
100,
and
250
mM NaC1). The reaction products were
analyzed using denaturing agarose gels. Fig.
5
shows a com-
parison of the reaction products obtained for the
100
mM
NaCl reaction and both pH
6
and pH
8.
At pH
6,
the size of
the DNA fragments obtained is much smaller than for the
same reaction performed at pH
8.
A
qualitatively similar effect
was observed for the
25
mM NaCl reactions (data not shown).
There is no observable effect seen for the
0
and
250
mM NaCl
concentrations. This is due to the mechanistic limitations
placed on the reactions by the extreme ionic conditions which
cannot be modulated by changes in pH (data not shown).
Analysis
of
AP Endonuclease Activity Utilizing Methoxy-
amine-In addition to the above analysis, aliquots from the
same time courses were terminated with SDS and treated
with
100
mM methoxyamine which has the effect of rendering
any apyrimidinic sites impervious to alkaline hydrolysis (34).
This allows the DNA fragment size generated by the AP
endonuclease activity to be analyzed during denaturing aga-
rose gels. Fig.
6
shows side-by-side identical time points in
the presence and absence of methoxyamine for both the pH
6
and pH
8
reactions. It can be clearly seen for both the pH
6
and pH
8
reactions that many AP sites are being resolved
by the alkaline treatment in the absence of methoxyamine
and that these AP sites must be the result of the enzyme
making only the glycosylic bond cleavage at the site of a
dimer. These data also suggest that the AP endonuclease has
a pH optimum around
6.
This effect was observed for all NaCl
concentrations tested (data not shown).
DISCUSSION
Previous work (37) had suggested that the glycosylase ac-
tivity of M. luteus UV endonuclease is more active than the
associated AP endonucleolytic activity in uitro. Our results
strongly support this observation as shown by the differences
17426
DNA Scanning Activity
of
M.
luteus
UV
Endonuclease
Panel
A
80
100
80
60
40
20
0
4
5
6
7
8
9
10
PH
Panel
B
4
5
6
7
8
9
10
PH
FIG. 4.
Determination
of
the pH optimum
for
the glycosy-
lase
and
AP
endonuclease activities
of
M.
luteus
UV
endonu-
clease.
In order to investigate the pH optimum for the enzyme,
reactions similar to those described in Fig.
1
were performed in the
following buffers at the pH values indicated by parentheses: Na+-
acetate (4.5, 5.0). MES (5.0, 5.5, 6.0), PIPES (6.0, 6.5), MOPS
(6.5,
7.0),
HEPES
(7.0,
7.5), Tricine
(7.5,
8.0, 8.5), CHES (8.5,
9.0),
and
CAPS
(9.0).
The final concentration for these buffers was
50
mM,
and the ionic strength of the reactions was
100
mM NaCI. Single time
points were taken after
20
min and terminated with and without the
alkaline nondenaturing treatment. These reaction products were then
analyzed by agarose gel electrophoresis and liquid scintillation count-
ing.
Panel
A
(form
I
DNA):
0,
(-)-alkaline treatment;
.,
(+)-alkaline
treatment.
Panel
R
(form
I11
DNA):
0,
(-)-alkaline treatment;
B,
(+)-alkaline treatment.
in the native and alkaline-treated time course reactions (see
Figs.
1,3,
and 6). We suggest that this is due to the apparent
lability of the AP endonuclease over time. This is very similar
to what has been observed for endonuclease V
(31,33)
where
the AP endonuclease displays
a
greater fragility than the
glycosylase activity. In addition to the apparent lability of the
AP endonuclease, our results suggest that this activity has
a
pH
6
lOOmM
pH
8
lOOmM
time (min)
0
5
10
15
20
30
time (min)
0
5
10
15
20
30
FIG.
5.
Kinetic analysis
of
M.
luteus
UV
endonuclease
at
pH
6
and
8.
Time course reactions similar to those described in Fig.
1
were performed using either 20 mM MES (pH 6.0) or
20
mM Tricine
(pH 8.0) and the indicated NaCl concentration. The reaction products
were analyzed using denaturing agarose gels.
pH optimum around 6.0,
as
shown by the methoxyamine-
treated reactions in Fig. 6,
as
well
as
the peak of form
I
loss
observed for the nonalkaline-treated reactions shown in
panel
A,
Fig. 4. The glycosylase activity,
as
judged by the loss of
form
I
DNA after alkaline hydrolysis
(panel
A,
Fig. 4), appears
to have a fairly wide pH optimum of 6.0-8.0. These are both
very consistent with the pH optimum observed for endonu-
clease V, 6.0-8.5 for the glycosylase, and
a
more narrow
optimum of around 6 for the AP endonuclease
(33).
Taken
together, these results illustrate even further the many simi-
larities between the T4 endonuclease V and the
M.
luteus
UV
endonuclease.
Our results support the following model for the DNA scan-
ning ability of
M.
luteus
UV endonuclease on UV-irradiated
DNA
in
uitro.
For reaction conditions which enhance protein-
DNA interactions
(0-25
mM NaCl (pH 8.0) or 0-100 mM
NaCl (pH 6.0), UV endonuclease binds to non-target DNA
and is able to diffuse along this DNA, until it encounters
a
pyrimidine dimer. After binding to that site, the enzyme
makes
a
glycosylase cut in the DNA
at
the site of the dimer.
The enzyme may either catalyze the phosphodiester bond
scission, then continue to scan additional non-target DNA,
or it may be released from the dimer site without completing
the phosphodiester bond scission thus leaving the alkaline-
labile site. The enzyme continues to scan the remaining DNA
for any additional dimers. Eventually all alkaline-labile sites
are converted to single-stranded breaks by the enzyme. How-
ever, from these data, we are unable to directly show that this
secondary AP endonuclease activity is acting in
a
processive
manner. Thus, depending on the lability
of
the AP endonu-
clease activity, many single-stranded breaks might require
a
secondary encounter with
a
dimer site after initially making
the glycosylic cleavage before the AP endonuclease cleavage
can be made. However, the analysis of the glycosylase activity
shows the enzyme to be scanning non-target DNA. At low
(0
and
25
mM NaCl) ionic strength conditions, the enzyme is
able to remain associated with
a
given plasmid long enough
for all glycosylic bond scissions to be made
at
the dimer sites.
At higher (100 and
250
mM NaCl) salt concentrations, espe-
cially at pH
8,
the increased ionic strength probably has
increased the dissociation constant of the enzyme with non-
DNA Scanning Activity
of
M.
luteus
UV
Endonuclease
17427
pH
6
lOOmM
(-)
methoxyamine
0
5
10 15
20
30
time (min)
I1
I
pH
8
lOOmM
(-)
methoxyamine
0
5
10
15
20
30
time
(min)
pH
6
lOOmM
(+)
methoxyamine
time (min)
0
5
10 15
20
30
The smaller fragment size obtained at pH
6
suggests that the
enzyme is acting sequentially
at
adjacent dimer sites within
the population of DNA. If the dissociation rate of the enzyme
for non-target DNA is significantly less
at
pH
6
than at pH
8,
the enzyme would remain bound longer and have
a
higher
probability of diffusing
a
greater distance along the DNA to
act at adjacent dimer sites; the enzyme would be acting more
processively. Thus, the pH of the reaction
in vitro
appears to
modulate the degree of processivity exhibited by the
M.
luteus
UV
endonuclease. We hypothesize that this effect may be due
to the titration of certain amino acid residues of the enzyme,
possibly histidine, to a net positive charge. This increase in
the number of positively charged residues may result in an
overall increase in the affinity of the enzyme for the negatively
charged phosphodiester backbone of non-target DNA. This
hypothesis is supported by the mutagenesis data described
previously for endonuclease
V
(25).
To
our knowledge, this
pH-dependent modulation
of
processivity has not been de-
scribed for any other DNA scanning enzymes.
REFERENCES
1.
Ptashne, M.
(1986)
Nature
322,697-701
2.
Lohman,
T.
M.
(1986)
CRC
Crit.
Reu.
Biochem.
19,191-245
3.
von Hippel,
P.
H., and Berg,
0.
G.
(1989)
J.
Rhl.
Chem.
264,675-678
(+)
methoxyamine
4.
Riggs, A.
D.,
Bourgeois,
S.,
and Cohn,
M.
(1970)
J.
Mol.
Riol.
53,401-417
5.
Berg,
0.
G.,
Winter, R. B., and von Hippel,
P.
H.
(1981)
Blochemutry
20,
15
2o
3o
6.
Berg,
0.
G., Winter, R. B., and von Hippel,
P.
H.
(1982)
Trends
Biochem.
7.
Winter, R. B., and von Hippel,
P.
H.
(1981)
Biochemistry
20.6948-6960
8.
Winter, R. B., Berg,
0.
G.,
and von Hippel,
P.
H.
(1981)
Biochemistry
20,
9.
Barkley,
M.
D.
(1981)
Biochemistry
20,3833-3842
pH
8
lOOmM
time (min)
fi929-6948
Sci.
7.52-55
6961-6977
10.
Llovd.
R.
S..
Hanawalt.
P.
C.. and Dodson. M. L.
(1980)
Nucleic Acids
Res.
FIG.
6.
Kinetic analysis
of
M.
luteus
UV
endonuclease
(2)-
methoxyamine.
Reactions identical with those described for Fig.
5
were performed. After terminating the reactions, methoxyamine
(100
mM) was added to render any remaining apyrimidinic sites impervious
to alkaline hydrolysis. Reactions were performed at the indicated pH
and NaCl concentrations. The reaction products were analyzed using
denaturing agarose gels.
target DNA to the point where it does not remain associated
with a plasmid long enough for all dimers to be completely
incised. Instead, the enzyme acts by
a
more distributive or
random hit mechanism in which the length of non-target
DNA that it is able scan before dissociating is shorter than
the interdimer distance for that plasmid. We also propose
that this dissociation rate can be modulated
in
vitro
by
changes in the pH of the reaction. This is clearly shown by
the difference in the size of the DNA fragments generated by
the enzyme at pH
6
versus
pH
8
at
100
mM NaCl (see Fig.
5).
11.
12.
13.
14.
15.
16.
17.
18.
8,
g113-5127
Biochemistry
25,5751-5755
Ganesan, A.
K.,
Seawell,
P.
C., Lewis, R.
J.,
and Hanawalt,
P.
C.
(1986)
Jack. W.
E..
Terrv. B.
J..
and Modrich.
P.
(1982)
Proc.
Natf.
Acad. Sci.
U.
Gruskin,
E.
A., and Lloyd, R.
S.
(1986)
J.
Biof.
Chem.
261,9607-9613
S.
A.
79,'4010:4014
'
Lansowski.
J..
Alves.
J..
Pineuod.
A..
and Maass.
G.
(1983)
Nucleic Acids
~
Res.
11,'50i-510
Ehhrecht, H.-J., Pinguod,
A.,
Urbanke, C., Maass,
G.,
and Gualeni, C.
Nardone,
G.,
George,
J.,
and Chirkjian,
J.
G.
(1986)
J.
Biof.
Chem.
261,
Belinstev, B.
N.,
Zauriev,
S.
K.,
and Shemyakin. M.
F.
(1980)
Nucleic Acids
Hannon, R., Richards,
E.
G.,
and Gald, H.
J.
(1980)
EMBO
J.
5, 3313-
..
.,
..
...
(1985)
J.
Riol.
Chem.
260,6160-6166
12128-12133
Res.
8,1391-1403
X119
19.
ParkiC.
S.,
Wu,
F.
Y.-H., and Wu, C.-W.
(1982)
J.
Biof.
Chem.
257,6950-
20.
Roe, J.-H., and Record, M.
T.,
Jr.
(1985)
Biochemistry
24,4721-4726
21.
Singer, P., and Wu, C.-W.
(1987)
J.
Riol.
Chem.
262.14178-14189
22.
Singer,
P.
T.,
and Wu, C.-W.
(19M)
J.
Biol.
Chem.
263,4208-4214
23.
Wang,
J.
C., and Giaever,
G.
N.
(1988)
Science
240,300-304
24.
Gruskin,
E.
A,,
and Lloyd, R.
S.
(1988)
J.
Riol.
Chem.
263, 12728-12737
25. Dowd,
D.
R., and Lloyd, R.
S.
(1989)
J.
Mol.
Riol.,
in press
26.
Haseltine, W. A,, Gorden, L.
K.,
Lundan, C. P., Grafstrom.
R.
H., Shaper,
27.
Gordon, L.
K.,
and Haseltine,
W.
A.
(1980)
J.
Rtof.
Chem.
255, 12047-
29.
Seawell,
P.
C., Smith, C. A,, and Ganesen, A.
K.
(1980)
J.
Virol.
35, 790-
28.
Radany,
E.
H., and Friedberg,
E.
C.
(1980)
Nature
286,182-185
30.
McMillan,
S.,
Edenberg, H. J., Radany,
E.
H., Friedberg, R. C., and
31.
Nakaheuou.
Y..
and Sekieuchi.
M.
(1981)
Proc. Natf. Acad. Sci.
U.
S.
A.
6056
N.
L., and Grossman, L.
(1980)
Nature
285,634-641
12050
797
Friedberg.
E.
C.
(1981)
J.
Virof.
40,211-223
78,2'7i212746
204-210
257,2556-2562
.,
.
32.
Warner, H. R., Christensen, L. M., and Persson, M.-L.
(1981)
J.
Virof.
40,
33.
Nakabeppu, Y., Yamashita,
K.,
and Sekiguchi, M.
(1982)
J.
Biol.
Chem.
34.
Liuzzi,
M.,
Weinfield, M., and Paterson, M. C.
(1987)
Biochemistry
26,
35.
Southern,
E.
M.
(1975)
J.
Mol.
Biof.
98,503-517
36.
Wahl,
G.
M., Stern, M., and Stark,
G.
K.
(1975)
Proc.
Natf. Acad. Sci.
U.
37.
Grafstrom, R. H., Park, L., and Grossman, L.
(1982)
J.
Biof.
Chem.
257,
:3:115-:3321
S.
A.
76,3683-3687
13465-13474
