Purifications of Micrococcus luteus UV damage-specific
or pyrimidine dimer-specific nicking activity have resulted in the
isolation of UV endonuclease proteins with molecular masses ranging
from 11 to 18
kDa(2, 3, 4, 5, 6) .
Haseltine et al.(7) proposed that strand scission at
a pyrimidine dimer required two activities, an N-glycosylase
and an apurinic/apyrimidinic (AP) (
)endonuclease. The UV
endonuclease-associated N-glycosylase activity would cleave
the N-glycosylic bond between the 5` pyrimidine partner of the
dimer and the corresponding deoxyribose moiety. Subsequently, an
independent AP endonuclease activity would cleave the sugar-phosphate
backbone on the 3` side of the apyrimidinic sugar moiety. Grafstrom and
co-workers (2) modified this proposal by suggesting that the N-glycosylase and AP endonuclease activities both reside on
the same UV endonuclease molecule. Characterization of an 18-kDa
protein has shown that the UV endonuclease prefers thymine-containing
dimers over cytosine-containing dimers (under conditions of substrate
excess), double-stranded over single-stranded DNA, and apyrimidinic
sites at the site of glycosylase action to simple apurinic or
apyrimidinic residues(2) . The M. luteus UV
endonuclease locates pyrimidine dimers, at least in vitro, by
a processive sliding mechanism on nontarget DNA(8) . The
efficiency of this scanning is dependent on both ionic strength and pH.
The 3` terminus generated by UV endonuclease requires further
processing by a class II endonuclease before DNA polymerase I and
ligase can seal the gap.
The catalytic mechanism of M. luteus UV endonuclease resembles those of a number of other enzymes that
perform the initial incision step of base excision repair;
bacteriophage T4 endonuclease V (1) , Saccharomyces
cerevisiae UV endonuclease(9) , Escherichia coli endonuclease III(10, 11, 12) , and E. coli Fpg (13, 14) also possess both N-glycosylase and AP lyase activities. Of these functionally
related enzymes, our laboratory is most familiar with the reaction
mechanism of T4 endonuclease V. T4 endonuclease V employs its
NH
-terminal,
-amino group in a nucleophilic attack of
the C-1` sugar carbon of the 5` nucleotide within the
dimer(15, 16) . The existence of the resultant imino
or Schiff base intermediate can be verified experimentally by
demonstrating both that cyanide can inhibit the enzyme in a
substrate-dependent manner (17, 18) and that
NaBH
can reduce the imino intermediate to a covalent
enzyme-substrate product(19) . The imino intermediate may or
may not undergo a subsequent
-elimination reaction that follows a syn stereochemical course to generate a trans 3`-
,
-unsaturated aldehyde and a 5`-phosphate
product(20, 21, 22) . Endonuclease III and
Fpg also are believed to utilize primary amino groups to form imino
intermediates(23, 24) , possibly Lys
in
the case of endonuclease III (25) and a primary amino group at
or near the amino terminus in the case of Fpg(24) . The AP
lyase steps of the M. luteus UV endonuclease(21) ,
endonuclease III(23, 26, 27) , and Fpg
enzymes all are known to proceed via
-elimination. The
-elimination step of endonuclease III, like that of T4
endonuclease V, follows a syn stereochemical course. Thus, a
unified catalytic mechanism for the N-glycosylase/AP lyases
has emerged; this family of enzymes employs a primary amine nucleophile
in its attack on the C-1` sugar carbon of the damaged nucleoside and,
in doing so, creates an identifiable imino
intermediate(28, 29) . Accordingly, the active site
residue of the M. luteus UV endonuclease also is hypothesized
to be a primary amine.
Since the M. luteus UV endonuclease
protein is established in the literature as an 18-kDa protein, it has
been compared most frequently with the 16-kDa T4 endonuclease V. This
report will demonstrate, however, that the M. luteus enzyme is
actually a 31- or 32-kDa protein; a prominent 18-kDa contaminant
copurified with UV endonuclease enzyme throughout many chromatographic
steps but was inactive against UV-damaged DNA. Moreover, cloning of the pdg (pyrimidine dimer glycosylase) gene revealed that the UV
endonuclease shares extensive sequence homology with the E. coli repair proteins endonuclease III and MutY, not T4 endonuclease V.
In addition to describing the isolation and cloning of M. luteus UV endonuclease, or more accurately pyrimidine dimer N-glycosylase/AP lyase, we will discuss characterization of
the purified enzyme and a preliminary investigation into its reaction
mechanism.
EXPERIMENTAL PROCEDURES
Materials
Lyophilized M. luteus cells (ATCC 4698) and lysozyme
were obtained from Sigma. The Sephadex G-100, phenyl-Sepharose CL-4B,
SP-Sepharose Fast Flow, Mono S, and Mono P matrices or columns were
purchased from Pharmacia Biotech Inc.; the Affi-Gel Blue and
heparin-agarose matrices were from Bio-Rad. Protein molecular weight
markers were purchased individually from Sigma or prestained, low
molecular size marker sets from Life Technologies, Inc. were employed.
Polyvinylidene difluoride membrane was obtained from Bio-Rad, and
microsequencing was carried out in the Protein Sequencing Core
Laboratory at Vanderbilt University.
[
-
P]ATP (3,000 Ci/mmol) was purchased from
DuPont NEN. New England Biolabs was the supplier for the T4
polynucleotide kinase and double-stranded M13mp18 and M13mp19 DNAs. The
oligonucleotides used in cloning were ordered from Research Genetics
(Huntsville, AL) or synthesized in the core facilities of Vanderbilt
University or the Sealy Center for Molecular Science. R408 helper
phage, E. coli XL1-Blue and LE392 cells, and an M. luteus genomic
ZAP II library were purchased from Stratagene. The
Sequenase version 2.0 DNA sequencing kit and M13 primers were obtained
from the U. S. Biochemical Corp. pBR322 DNA was produced in our
laboratory or by the Sealy Center Recombinant DNA Laboratory. All
49-mer, dimer-containing oligonucleotides were provided generously by
John-Stephen Taylor and Colin Smith (Washington University, St. Louis,
MO). The 37-mer, 5,6-dihydrouracil-containing oligonucleotide and its
complement were the gift of Paul Doetsch and L. Augeri (Emory
University, Atlanta, GA). Oligonucleotide sizing markers were purchased
from Pharmacia. NaBH
and piperidine stocks were Sigma
products. NaCN was obtained from Fisher Scientific Co.
Purification Scheme 1
Isolation of the 18-kDa Ribosomal
Protein
All purification procedures were performed at 4
°C. Thirty g of lyophilized M. luteus cells was suspended
in 1,500 ml of 20 mM Tris
HCl, pH 7.5, 10 mM EDTA, 200 mM KCl (buffer A), allowed to hydrate for
several hours, and then lysed overnight by the addition of lysozyme at
600 µg/ml into buffer A. The following day, the viscous mixture was
sonicated with a Branson Sonifier 450 until its consistency thinned, an
indication that the majority of the cells had been disrupted.
Microscopic examination of the pre- and postsonication samples also
confirmed that essentially complete lysis had been achieved. The
cellular debris was pelleted by centrifugation at 13,000
g for 1 h, and the resultant supernatant was loaded onto a
single-stranded DNA-agarose column (20 cm
75 cm)
that had been equilibrated with buffer A containing 10% (v/v) ethylene
glycol(30) . After the column was loaded and washed thoroughly
with the equilibration buffer, bound proteins were eluted with a
2-liter linear gradient of equilibration buffer containing
200-2,000 mM KCl. Eleven-ml fractions were collected and
monitored for M. luteus UV endonuclease activity, as in all
subsequent purification steps and schemes, by assaying their ability to
nick UV-irradiated pBR322 DNA and/or thymine dimer-containing,
site-specific oligonucleotides. Although fractions were also examined
by SDS-PAGE coupled to either Coomassie or silver staining, they were
pooled according to their activity rather than their molecular mass
profiles. Single-stranded DNA-agarose fractions 91-141 were
combined (1,100-1,500 mM KCl, 600 ml), concentrated to
200 ml on an Amicon concentrator equipped with a YM10 membrane under 40
p.s.i. of pressure, and loaded in two batches of 100 ml each onto a
Sephadex G-100 (20 cm
100 cm) column that had been
equilibrated with 25 mM Na
HPO
, pH 7.5,
1 mM EDTA, 100 mM KCl, 10% (v/v) ethylene glycol
(buffer B). During each run, 13-ml fractions were collected, and the
majority of the UV damage-specific nicking activity was found in
fractions 41-61. The active fractions from both runs (450 ml)
were applied to a heparin-agarose column (3.0 cm
10
cm) that had been equilibrated with buffer B. After thorough washing of
the column with equilibration buffer, elution with a 500-ml linear
gradient of 100-400 mM KCl in buffer B resulted in the
enzyme activity peaking between 11-ml fractions 8 and 26 (150-270
mM KCl, 209 ml). This material, once diluted 2-fold with
buffer B minus KCl, was reloaded onto the reequilibrated
heparin-agarose column and eluted a second time with a 100-250
mM KCl, 500-ml linear gradient of buffer B. Twelve-ml
fractions 2-15 (110-150 mM KCl, 168 ml) contained
the majority of the UV endonuclease activity and were pooled for
further purification on a small single-stranded DNA-agarose column (1.8
cm
50 cm). The sample was loaded, the column was
washed with buffer B, and a 500-ml linear gradient of 100-1,500
mM KCl in buffer B was run. Fraction 23 (850 mM, 11.5
ml) was prepared for phenyl-Sepharose chromatography by slowly adding
(NH
)
SO
to a final concentration of
1 M. The phenyl-Sepharose column (3.0 cm
7.0 cm) was equilibrated with Na
HPO
, pH 7.5, 1
mM EDTA, 1,000 mM
(NH
)
SO
, 10% (v/v) ethylene glycol
(buffer C), loaded, washed briefly with its high salt equilibration
buffer C, and then eluted of proteins with a 200-ml gradient of
1,000-0 mM (NH
)
SO
buffer C. Four-ml fractions 31-41 (380-180
mM (NH
)
SO
, 44 ml) were
combined, concentrated through Amicon filtration to 10 ml, diluted with
50 ml of 10 mM Tris
HCl, pH 7.5, 1 mM EDTA to
lower the salt concentration, and again concentrated, this time to 7
ml. Concomitantly, 10 ml of small single-stranded DNA-agarose fraction
24 was diluted with 130 ml of 10 mM Tris
HCl, pH 7.5, 1
mM EDTA prior to concentration to 10 ml. Each sample was then
ready to be loaded onto an FPLC Mono S column that had been
equilibrated with 10 mM Tris
HCl, pH 7.5, 10 mM NaCl. In the first Mono S run, the phenyl-Sepharose sample was
loaded, the column was washed briefly with equilibration buffer, and a
linear gradient of 10 mM Tris
HCl, pH 7.5, buffer
containing 10-750 mM NaCl was programmed and executed.
The majority of the enzyme activity was contained within 1.5-ml
fractions 13-16 (6 ml). The second Mono S run (single-stranded
DNA-agarose sample) was conducted following the same protocol with UV
damage-specific nicking activity eluting primarily in fractions
13-18 (12 ml). Side fractions 8 and 9 from the first Mono S run
and 11-13 from the second Mono S run were pooled, at this
particular step, according to SDS-PAGE silver stain results; the peaks
for the 18-kDa protein and UV damage-specific nicking activity were by
now somewhat skewed. The (NH
)
SO
concentration of the sample was raised to 1 M, and the
Mono S material was applied once more to the phenyl-Sepharose column
that had been equilibrated in buffer C minus ethylene glycol. After the
column had been washed thoroughly, the 18-kDa protein was eluted with a
200-ml linear gradient of equilibration buffer containing 1,000-0
mM (NH
)
SO
. Fractions 37
and 38 were combined (6 ml), mixed with an equal volume of 20% (w/v)
trichloroacetic acid, set on ice for 30 min to precipitate, and
subjected to electrophoresis on a 13% SDS-PAGE gel. The 18-kDa protein
(30-50 pmol) was transferred to polyvinylidene difluoride paper
according to the method of Matsudaira(31) , excised from the
membrane, and microsequenced via Edman degradation.
Cloning of the 18-kDa Ribosomal
Protein
Based on the amino-terminal sequence of the 18-kDa
protein, sense and antisense 42-mer primers were designed for cloning
of the protein: 5`-ATGTCCCGCATCGGCCGCCTCCCGATCACCATCCCGGCCGGC-3` (852)
and 5`-GCCGGCCGGGATGGTGATCGGGAGGCGGCCGATGCGGGACAT-3` (853). Southern
blots performed on restriction digest gels of M. luteus genomic DNA (prepared from the freeze-dried cells) both confirmed
that a signal could be detected using either probe and helped to
optimize hybridization conditions for the actual cloning procedure. As
the initial step in the cloning strategy, a randomly sheared M.
luteus genomic
ZAP II library was plated on NZY (1% (w/v) NZ
amine, 5% (w/v) NaCl, 2% (w/v) MgSO
7H
O)
+ 0.7% agarose-overlaid plates using E. coli LE392 as the
host bacterium. Plaques were visible after the bacteriophage had grown
overnight at 37 °C, so the plates were chilled for 1 h prior to
lifting. The nitrocellulose filters were processed successively in 30-s
steps with solutions A (1.5 M NaCl, 0.5 M NaOH), B
(0.5 M Tris
HCl, pH 7.5), and C (3 M NaCl, 300
mM sodium citrate, pH 7.0) to denature the DNA. The filters
were baked in a vacuum oven for 2 h at 80 °C and then immersed in
prehybridization solution (20% formamide, 5
SSPE where 1
SSPE equals 50 mM
NaH
PO
H
O, pH 7.4, 750 mM NaCl, 5 mM EDTA), 5
Denhardt's solution,
100 µg/ml fish milt DNA, 0.1% SDS) for 4 h at 48 °C.
P-Labeled probe 853 was added, and the hybridization was
allowed to proceed overnight at 48 °C. After a quick rinse, the
filters were washed twice for 30 min with 4
SSPE (200
mM NaH
PO
H
0, pH 7.4,
600 mM NaCl, 20 mM EDTA), dried, and exposed to XAR-5
film (Kodak) at -70 °C. Eight positive plaques were detected,
cored, and eluted into suspension medium (50 mM Tris
HCl,
pH 7.5, 100 mM NaCl, 8 mM
MgSO
7H
O, 0.01% (w/v) gelatin). Two
different concentrations of each eluate were replated for a second
round of screening. Plates for each positive were screened with either
probe 852 or 853, and only clones of 1, 2, and 6 survived this second
generation hybridization. Again, the positive plaques were cored and
eluted into suspension medium with gentle shaking. In addition to being
replated for a third round of screening, the positive phage were
rescued as phagemid in the following manner. Core eluates were
incubated with log phase E. coli XL1-Blue cells and R408
helper phage for 15 min at 37 °C. Each cell mixture was then added
to 5 ml of 2
YT (16% (w/v) Bacto-tryptone, 10% (w/v)
Bacto-yeast extract, 5% (w/v) NaCl, pH 7.0) medium and allowed to grow
for 3 h at 37 °C. The cells were lysed by heating the tubes for 20
min at 70 °C and centrifuged at 4,000
g for 5 min.
pBluescript phagemid packaged as filamentous phage particles were
recovered in the supernatant and plated with log phase E. coli XL1-Blue cells onto 10 Luria Bertani plates supplemented with 50
µg/ml ampicillin. Meanwhile, in the third and final round of
screening, plaques derived from original clones 1 and 6 hybridized to
probe 852; clone 2 proved to have been a false positive. Single
colonies were picked from the XL1-Blue plates, cultures grown
overnight, and DNA isolated using a STET (10 mM Tris
HCl,
pH 8.0, 100 mM NaCl, 1 mM EDTA, 5% (v/v) Triton
X-100) preparation procedure. A series of restriction enzyme
digests/Southern blots suggested that the M. luteus gene could
be subcloned into M13mp18 and M13mp19 using a SacI/EcoRI ligation strategy. The ligation mixtures
were transformed into E. coli UT481 bacteria, the plaques were
probed with 852 and 853, and positives were cored and grown overnight
in 10 ml of 2
YT at 37 °C. While replicative form DNA was
harvested from the culture pellets, single-stranded M13 DNA was
recovered from the supernatants. Sequencing focused on the
M13mp18-clone 1 and M13mp19-clone 1 constructs. The M13 universal
primer was used in the initial stages of sequencing, but, as sequencing
progressed throughout the gene, it became necessary to design
additional primers. Homology searching was done using the National
Center for Biotechnology Information Blaster program.
Purification Scheme 2
Isolation of the 31/32-kDa M. luteus UV
Endonuclease
Many of the experimental details are parallel
to those presented in purification scheme 1 and can be inferred from
the above protocol. Fifty-four g of M. luteus cells was
suspended in 2,500 ml of buffer A and allowed to hydrate overnight.
Next, lysozyme was added into the cell slurry to a final concentration
of 400 µg/ml, and lysis was allowed to proceed until the following
day. The concentration of KCl in buffer A was elevated to 400 mM prior to sonication to accelerate mechanical disruption of the
cells. Two 30-min centrifugation spins at 2,600
g were
required to obtain a particle-free supernatant that could be loaded
onto a single-stranded DNA-agarose column (20 cm
75
cm). Before loading, however, the cellular extract was diluted with an
equal volume (3 liters) of 20 mM Tris
HCl, pH 7.5, 10
mM EDTA to return the salt concentration to 200 mM.
Thirteen-ml fractions were collected from a 2-liter gradient of buffer
A containing 200-2,000 mM KCl. Fractions 56-106
(850-1,400 mM, 700 ml) were pooled and applied to an
Affi-Gel Blue column (5.0 cm
10 cm) that had been
equilibrated with buffer A containing 500 mM KCl. The UV
endonuclease enzyme bound weakly to the column and emerged in the
flow-through, wash, and 8-ml fractions 1-11 (500-1,500
mM KCl) of a 300-ml, 500-4,000 mM buffer A
gradient. The active effluents were combined (950 ml), diluted 2-fold
with 20 mM Tris
HCl, pH 7.5, 10 mM EDTA,
reloaded onto the Affi-Gel Blue column, and the proteins again eluted
with a 300-ml, 500-4,000 mM buffer A gradient. This
time, the flow-through, wash, and fractions 1-21 (500-2,500
mM KCl) were pooled (2,100 ml) in preparation for loading onto
a buffer C-equilibrated phenyl-Sepharose column (3.0 cm
7.0 cm). Initially, in a trial run, 525 ml of the
2,100-ml Affi-Gel Blue pool was loaded onto the phenyl-Sepharose
column, and the remainder was applied in a second run. Before each run,
(NH
)
SO
was slowly mixed into the
samples to achieve a final concentration of 1 M, and a 200-ml
gradient of 1,000-0 mM
(NH
)
SO
in buffer B was employed to
elute the UV damage-specific nicking activity. Five-ml fractions
25-50 (130 ml) from the first phenyl-Sepharose run and 4-ml
fractions 5-29 (100 ml) from the second phenyl-Sepharose run were
pooled independently. An additional 25 mM
NaH
PO
, pH 6.8, was added to each pool before it
was applied to a Sephadex G-100 column (20 cm
100
cm) that had been equilibrated in 25 mM
NaH
PO
, pH 6.8, 1 mM EDTA, 100 mM KCl, 10% (v/v) ethylene glycol (buffer D). In each run, as soon as
the sample was loaded, buffer D was loaded onto the column, and 12-ml
fractions were collected. Fractions 26-66 from the first Sephadex
G-100 run (first phenyl-Sepharose run material) and fractions
41-56 from the second Sephadex G-100 run (second phenyl-Sepharose
run material) were combined, and 150 ml of the 600-ml pool was applied
to a heparin-agarose column (5.0 cm
7.0 cm) that
had been equilibrated with buffer D. After the column was washed
thoroughly with equilibration buffer, elution with a 150-ml linear
gradient of 100-400 mM KCl in buffer D yielded an enzyme
activity peak in 5-ml fractions 33-38 (30 ml). This pool, along
with the remainder of the Sephadex G-100 pool, was loaded onto a
UV-irradiated single-stranded DNA-agarose column (0.5 cm
30 cm) that had been equilibrated with buffer B
containing 200 mM KCl. Proteins that remained bound after
extensive washing were eluted with a 1-liter linear gradient of
200-1,500 mM KCl in buffer B. Eleven-ml fractions
21-41 (500-800 mM KCl, 230 ml) were combined and
diluted with an equal volume of 10 mM Tris
HCl, pH 8.0, 1
mM EDTA. This starting material was batch loaded onto the
Affi-Gel Blue matrix that had been batch equilibrated with buffer B
containing 200 mM KCl. UV endonuclease activity eluted in 7-ml
fractions 16-33 (1050-2,000 mM KCl, 126 ml) of a
500-ml linear gradient of 200-4,000 mM buffer B. Five ml
of fraction 19 and 6 ml of 20, 21, or 22 were dialyzed separately
against 25 mM NaH
PO
, pH 6.8, 100
mM NaCl (buffer E). Each sample was loaded onto an FPLC Mono S
column that had been equilibrated with buffer E, and a 38-ml gradient
of 100-500 mM NaCl in buffer E was executed. Nicking
activity eluted consistently in the 260-325 mM NaCl
range. Fraction 18 (1 ml) from the Affi-Gel Blue fraction 20 run was
dialyzed against 25 mM ethanolamine, pH 10.0, 10 mM NaCl and then injected onto an FPLC Mono P column that had been
equilibrated in the same buffer. UV endonuclease activity eluted in
1-ml fractions 3, 4, and 5 of a 38-ml gradient of equilibration buffer
containing 0-8% Pharmacia Polybuffer 96 (pH gradient
10.0-7.0). Mono S 1-ml fractions 17, 19, and 21 from the Affi-Gel
Blue fraction 19 run, 15-17 and 19-21 from the Affi-Gel
Blue fraction 20 run, 14-19 from the Affi-Gel Blue fraction 21
run, and 16, 17, 19, and 20 from the Affi-Gel Blue fraction 22 run were
pooled independently and precipitated with a sixth volume of 70% (w/v)
trichloroacetic acid. The samples were subjected to electrophoresis on
a 13% SDS-PAGE gel, the 31- and 32-kDa proteins (20-40 pmol each)
were transferred to polyvinylidene difluoride membrane, carefully
excised, and microsequenced via Edman degradation.
Cloning of the 31/32-kDa M. luteus UV
Endonuclease
Based on the amino-terminal sequence of the
31- and 32-kDa proteins, the following sense and antisense 38-mer
primers were designed for cloning of the UV endonuclease gene:
5`-ATGGAGACGGAGTCCACGGGCACGCCGACGGGCGAGAC-3`(1083) and
5`-GTCTCGCCCGTCGGCGTGCCCGTGGACTCCGTCTCCAT-3`(1084). Southern blots were
performed on restriction digest gels of M. luteus genomic DNA
(prepared from freeze-dried cells) both to confirm that a signal could
be detected using either probe and to optimize hybridization conditions
for the actual cloning procedure. The randomly sheared M. luteus genomic
ZAP II library was plated and hybridized at 42 °C
with
P-labeled probe 1083. Two of the 10 positive clones
from the initial plating of the M. luteus library survived
three subsequent rounds of hybridization. These positives were rescued
as phagemid into pBluescript, the inserts were subcloned into M13mp18,
and the M13mp18-clone 3 construct was sequenced. Unfortunately,
although a 4-kilobase fragment of genomic DNA had been isolated, only
the first 666 bp of the pyrimidine dimer N-glycosylase/AP
lyase coding sequence were present. An additional 1.5
10
plaques were screened resulting in the isolation of a larger
5.5-kilobase genomic DNA fragment. Using probes spanning the length of
the known 666-bp sequence, a series of restriction digestions and
Southern blots was performed to determine the position of the gene of
interest within the 5.5-kilobase fragment. A 2.0-kilobase MluI
fragment thought to incorporate the full-length gene was isolated and
its overhang ends blunted so that it could be ligated into the SmaI site of M13mp18.The pdg gene was sequenced
with a battery of sense oligonucleotides and then subcloned into
M13mp19 or plasmids for sequencing in the opposite direction with
antisense primers. GC-rich stretches of the sequence often were
compressed or poorly resolved, thus making the exact sequence difficult
to interpret. Those regions that were recalcitrant to standard Sanger
dideoxy sequencing were resequenced with 7-deaza-2`-deoxy-GTP in the
deoxyribonucleoside triphosphate mix or by the Maxam-Gilbert method.
Alternatively, sequential and overlapping sections of the gene were
amplified by polymerase chain reaction and then sequenced individually.
Purification Scheme 3:
Twenty-five g of M. luteus cells was suspended in
1,250 ml of buffer B minus ethylene glycol, allowed to hydrate
overnight, and then lysed for the next 24 h by the addition of 400
µg/ml lysozyme into the cell slurry. The KCl concentration of the
slurry was raised to 300 mM immediately before sonication.
Cellular debris was pelleted by two 30-min rounds of centrifugation at
8,000
g, and the resultant supernatant was loaded onto
a single-stranded DNA-agarose column (20 cm
75 cm)
that had been equilibrated in buffer B. The majority of the UV
endonuclease activity eluted in 12-ml fractions 100-140
(890-1,300 mM KCl, 500 ml) of a 2-liter, 300-2,000
mM KCl buffer B gradient. In a trial run, fractions
128-132 (60 ml) were pooled, dialyzed against 25 mM NaH
PO
, pH 6.8, 1 mM EDTA (buffer
F), and applied to an SP-Sepharose column (1.8 cm
45 cm) that had been equilibrated in buffer F. A 500-ml linear gradient
of 0-800 mM KCl in buffer F concentrated enzyme activity
in 3.5-ml fractions 24-36 (5-70 mM KCl, 45 ml).
Since a limited number of candidate proteins could be detected in these
active fractions by silver staining, another SP-Sepharose column (1.8
cm
100 cm) was packed and equilibrated with buffer
F. Single-stranded DNA-agarose fractions 102-127 were pooled (312
ml), dialyzed against 15 mM Na
HPO
, pH
7.6, 1 mM EDTA (buffer G), and loaded onto the new matrix.
After the column was washed, it was eluted of proteins with a 600-ml
linear gradient of 0-400 mM KCl in buffer G. Although UV
endonuclease activity was spread out between fractions 60 and 100,
fractions 64-76 (220-250 mM KCl, 57 ml) were
pooled and combined with fractions 24-36 from the first
SP-Sepharose run (102 ml total volume).
(NH
)
SO
was added to a final
concentration of 1.8 M before the SP-Sepharose material was
loaded onto a phenyl-Sepharose column (1.8 cm
45
cm) that had been equilibrated with 25 mM
NaH
PO
, pH 6.8, 1.8 mM
(NH
)
SO
, 1 mM EDTA, 100
mM KCl, 10% (v/v) ethylene glycol (buffer H). A 400-ml linear
gradient of 1.8-0 M (NH
)
SO
in buffer H was run, but no nicking activity was found until 3-ml
fractions 180-290, fractions that were collected during a
postgradient wash with buffer H. In fact, UV endonuclease activity
continued to trail off in fractions 291-330 as the column was
being stripped with 25 mM NaH
PO
, pH
6.8, 1 mM EDTA, 10% ethylene glycol.
M.luteus UV Endonuclease UV Damage-specific Nicking
Activity on Plasmid DNA
In purification schemes 1-3, fractions were monitored
routinely for their ability to nick irradiated versus unirradiated plasmid DNA. pBR322 plasmid DNA was irradiated with
254-nm light at 100 microwatts/cm
for 245 s to generate
20-25 pyrimidine dimers/plasmid molecule(32) . The DNA
was then diluted from approximately 1 µg/µl to 0.100
µg/µl in 25 mM Na
HPO
, pH 7.5,
1 mM EDTA, 100 µg/ml bovine serum albumin (purification
schemes 1 and 2) or to 0.075 µg/µl in 25 mM NaH
PO
, pH 6.8, 25 mM NaCl, 1
mM EDTA, 100 µg/ml bovine serum albumin (purification
scheme 3). Varying amounts of the column fractions, usually 1-5
µl or dilutions thereof, were incubated with 20 µl of
irradiated or unirradiated pBR322 (2.0 µg of DNA in purification
schemes 1 and 2, 1.5 µg in purification scheme 3) for 30 min at 37
°C. Reactions were terminated by the addition of an equal volume of
electrophoresis loading buffer (50 mM Tris
HCl, pH 8.0,
20 mM EDTA, 40% (w/v) sucrose, 2% (w/v) SDS, 0.02% (w/v)
bromphenol blue, 0.02% (w/v) xylene cyanol). Form I (supercoiled), form
II (nicked circular), and form III (linear) DNAs were resolved by
electrophoresis through a 1% (w/v) agarose gel in 40 mM Tris
OAc, pH 8.0, 1 mM EDTA running buffer. The gel
was stained in 0.5 µg/ml ethidium bromide so that the three
topological forms of pBR322 could be visualized on a longwave UV
lightbox. To quantitate the data, images of the gels were captured by a
digital camera system (The Imager, Appligene) and then analyzed using
BioImage software (Millipore).
M. luteus UV Endonuclease Damage- and
Mismatch-specific Nicking Activities on Site-specific Oligonucleotide
Duplexes
In purification scheme 3, fractions were monitored for both
their ability to nick irradiated plasmid DNA and their ability to nick
a 49-bp oligonucleotide duplex containing a site-specific cis-syn thymine dimer. In addition, purified M. luteus UV
endonuclease (phenyl-Sepharose fraction 315) was tested for its ability
to nick 49-mers containing a trans-syn-I, ), or Dewar thymine
dimer(33) , a 37-mer containing a 5,6-dihydrouracil lesion, and
50-mers containing an A:G or A:C mismatch. The trans-syn-I,),
and Dewar thymine dimer-containing duplexes shared the following
sequence in the damaged strand:
5`-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3`. The cis-syn damaged strand had a slightly altered sequence:
5`-AGCTACCATGCCTGCACGTATTATGCAATTCGTAATCATGGTCATAGCT-3`. Each
dimer type bridged the two thymines in boldfaced positions 21 and 22 of
the 49-mers relative to their 5` ends. Control 49-mer incorporated the
AATTAA sequence but was left undamaged at the TT site.
The damaged strand of the 37-mer duplex had the sequence
5`-CTTGGACTGGATGTCGGCACXAGCGGATACAGGAGCA-3` with the X in position 21 representing a 5,6-dihydrouracil
lesion. The complementary strand incorporated a G opposite the X. In the 50-mer ``damaged'' strand,
5`-AGCTACCATGCCTGCACGAGATAAGCAATTCGTAATCATGGTCATAGCTA-3`, the A
at position 21 served as the mismatched base when it was paired with a
C or a G in the complement. The control complement incorporated a T
opposite the A. Each type of damage-containing strand was
P labeled on its 5` end with T4 polynucleotide kinase and
then annealed to its complement. Depending upon the experiment, varying
amounts of phenyl-Sepharose fraction 315 or control enzyme were
incubated at 37 °C for the indicated length of time with the
appropriate duplex 49-mer (0.4 ng = 12.5 fmol in a 10-µl
volume or 0.8 ng = 25 fmol in a 20-µl volume), 37-mer duplex
(0.8 ng = 32 fmol in a 20-µl volume), or 50-mer duplex (0.5
ng = 16 fmol in a 10-µl volume) in 25 mM NaH
PO
, pH 6.8, 1 mM EDTA, 100
µg/ml bovine serum albumin. Reactions were terminated either by
placing the reaction tubes in a dry ice-EtOH bath or by treating the
reaction mixtures with 1 M piperidine for 30 min at 85 °C.
When piperidine treatment was carried out, reaction samples were dried
down in a Savant SC110 SpeedVac and then resuspended in twice their
original reaction volume of loading buffer (95% (v/v) formamide, 20
mM EDTA, 0.02% (w/v) bromphenol blue, 0.02% (w/v) xylene
cyanol). When piperidine treatment was not carried out, an equal volume
of formamide loading buffer was added to the samples. All samples were
heated for 3-5 min at 80 °C prior to being loaded onto 15%
polyacrylamide gels containing 8 M urea. The oligonucleotide
bands were visualized by autoradiography of the wet gels with
Hyperfilm-MP film (Amersham Corp.) at -70 °C, typically for
several hours with two DuPont Quanta III intensifying screens enclosed
in the cassette. When it was necessary to obtain quantitative results,
the wet gels were also scanned on a PhosphorImager 450 machine
(Molecular Dynamics) and the data analyzed using Image Quant software
(Molecular Dynamics).
RESULTS
Purification and Cloning of the 18-kDa Ribosomal
Protein
The original intent of purification scheme 1 was to
isolate the 18-kDa M. luteus UV endonuclease that had been
reported in the literature(2) . Since the M. luteus UV
endonuclease was believed to be similar in structure to T4 endonuclease
V, the purification scheme incorporated many of the chromatographic
steps that are routinely employed by our laboratory to purify
endonuclease V: single-stranded DNA-agarose, Sephadex G-100,
heparin-agarose, phenyl-Sepharose, and FPLC Mono S(34) . Column
fractions were assayed for M. luteus UV endonuclease activity
by performing nicking assays on UV-irradiated versus nondamaged pBR322 plasmid DNA. The protein makeup of the fractions
was assessed via SDS-PAGE coupled to either Coomassie Blue or silver
staining. After following the UV damage-specific nicking activity of
the M. luteus lysate over five different column types, with
some columns being run twice, an 18-kDa protein emerged as the most
prominent band. Contaminants were still present even after the FPLC
Mono S runs, however, with a 31/32-kDa doublet being particularly
noticeable (Fig. 1A). Consequently, the Mono S
fractions were pooled according to protein content, and the 18-kDa
protein was purified further to
80% homogeneity on a second
phenyl-Sepharose column. Microsequencing of the first 14 amino acids of
the 18-kDa protein (Table 1) and knowledge of the unique codon
usage pattern of M. luteus(35, 36) enabled
us to design sense and antisense 42-mer primers to clone the potential
endonuclease V homolog. Cloning yielded a 713-bp sequence that spanned
the end of the S8 ribosomal protein gene, the open reading frame of the
L6 ribosomal protein gene, and the beginning of the L18 ribosomal
protein gene of the M. luteus spectinomycin
operon(35, 36, 37) . The amino acid sequence
of the 18-kDa protein clearly matched with that of the L6 ribosomal
protein (EMBL accession number X17524) and not a repair-related
protein; the NH
-terminal amino acid sequences were
identical, and our L6 gene sequence differed from the published
sequence in only 12 regions of minor sequence discrepancies.
Figure 1:
Purification schemes 1 and 2:
resolution of the 18-kDa ribosomal protein and the 31/32-kDa UV
endonuclease protein. Fraction 10 (2 ml) from the first Mono S run of
purification 1 was trichloroacetic acid precipitated and subjected to
electrophoresis on a 15% SDS-PAGE gel. Panel A shows the
silver stain of the appropriate lane of the gel. During purification
scheme 2, Mono P fractions 3, 4, and 5 (1 ml each) were trichloroacetic
acid precipitated and subjected to electrophoresis on a 15% SDS-PAGE
gel in lane 1, 2, or 3, respectively. Panel B shows the silver stain of the gel. The positions of
several molecular size markers are noted to the left of panels A and B: ovalbumin (45 kDa),
-lactoglobulin (18 kDa), and lysozyme (14 kDa). The arrows to the right of panels A and B point at
either the 31/32-kDa UV endonuclease or the 18-kDa ribosomal protein.
The UV damage-specific nicking activity associated with the fractions
shown in panel B, lanes 1-3, is presented in panel C, lanes 1-3. In the assay shown in panel C, enzyme (5 µl of each fraction) was incubated with
irradiated pBR322 DNA (2.0 µg) for 30 min at 37 °C. Form I
(supercoiled), form II (nicked circular), and form III (linear) DNAs
were resolved by electrophoresis through a 1% (w/v) agarose gel. The control lane (C) contained irradiated pBR322 plasmid
DNA that had been incubated but without enzyme. To quantitate the
silver stain or agarose gel bands, images of the gels were captured by
a digital camera system and then analyzed using BioImage
software.
Purification and Cloning of the 31/32-kDa M. luteus
UV Endonuclease
After concluding that the 18-kDa protein
was not the M. luteus UV endonuclease, the protein and
activity profiles collected during purification scheme 1 were
reexamined. Reinspection of the data showed that the peaks for the
18-kDa protein and UV damage-specific nicking activity had been
slightly offset; faint 31- and 32-kDa protein bands correlated more
directly with protein activity. Purification scheme 2 therefore was
undertaken with the purpose of isolating this protein doublet. It
included the following chromatographic steps: single-stranded
DNA-agarose, Affi-Gel Blue, phenyl-Sepharose, Sephadex G-100,
heparin-agarose, UV-irradiated single-stranded DNA-agarose, FPLC Mono
S, and FPLC Mono P. As was expected from the observations described
above, the UV damage-specific nicking activity was retained even after
the removal of small molecular weight proteins (M
11,000-18,000) from the enzyme preparation. Fig. 1, B and C, illustrates the correspondence between the
amount of 31/32-kDa protein and the level of UV damage-specific nicking
activity for several FPLC Mono P fractions. Quantitative analysis of Fig. 1B indicated that lane 1 (fraction 3)
contained 5.6 times as much protein at the 31- and 32-kDa positions as
did lane 2 (fraction 4), but no protein was detected in
lane 3 (fraction 5). Plasmid nicking assays with 5 µl of
each fraction generated the following form I:II:III product ratios: lane C (no enzyme), 45:55:0; lane 1 (fraction 3),
0:88:12; lane 2 (fraction 4), 13:85:2; and lane 3 (fraction 5), 42:58:0. Given the assay design, form III had been
expected to accumulate linearly proportional to enzyme
concentration(38) , so it followed that fraction 3 had produced
approximately six times as many double-stranded breaks per plasmid than
fraction 4. Identical NH
-terminal amino acid sequences were
obtained from microsequencing of the 31- and 32-kDa proteins (Table 1), and the common sequence was used to design sense and
antisense 38-mer primers.Cloning identified an 804-bp open reading
frame encoding a protein of 268 amino acids with a calculated molecular
mass of 29,306 Da (Fig. 2A). Curiously, a second stop
codon was present at bp position 838 which could potentially signal the
termination of a longer protein of 279 amino acids with a calculated
molecular mass of 30,340 Da. Although we were unsure of how the M.
luteus transcription machinery might bypass the first OPA codon,
perhaps through an amber suppressor-type mechanism, it seemed plausible
that the 268- and 279-residue proteins corresponded to the two forms of
purified UV endonuclease protein with apparent molecular masses of 31
and 32 kDa, respectively. Indeed, amino-terminal sequence data obtained
for the first 35 residues of the 31-kDa protein and the first 24
residues of the 32-kDa protein matched the NH
-terminal
sequence predicted by the cloned gene. Comparable to other M.
luteus genes that have been sequenced, pdg possessed an
overall GC content of 72%, and 94% of its codons contained a G or C in
the third position. Also, a purine-rich, Shine-Dalgarno-like sequence
was present 14 to 8 bases upstream of the start codon.
Figure 2:
DNA sequence of the pdg gene and
amino acid sequence alignment of UV endonuclease, endonuclease III, and
MutY. Panel A presents the nucleotide sequence of the pdg open reading frame and its flanking 5` and 3` sequences. The
deduced amino acid sequence of the pyrimidine dimer N-glycosylase/AP lyase protein is given above the DNA
sequence. The initiation and termination codons are shown in boldface type. A Shine-Dalgarno-like sequence is underlined. Panel B shows a comparison of the protein
sequences of the M. luteus pyrimidine dimer N-glycosylase/AP lyase and E. coli endonuclease III
and MutY DNA repair proteins. Regions of strong homology are indicated
by upper case as opposed to lower case lettering.
Alignments and homology assessments were determined initially using the
National Center for Biotechnology Information multiple alignment
construction and analysis workbench program (MACAW) and then edited
manually. Cysteines within the conserved
C-X
-C-X
-C-X
-C
coordination motif and the putative catalytic residues for endonuclease
III and UV endonuclease (Lys-120 and Lys-135, respectively) are highlighted with boldfaced type.
A GenBank
search revealed that the protein deduced from the pdg open
reading frame shared significant homology with two E. coli repair proteins, endonuclease III (31% identity across the length
of the M. luteus protein) and MutY (22% identity) (Fig. 2B), and an uncharacterized, pFV1 plasmid-encoded
protein from Methanobacterium thermoformicicum (the open
reading frame 10 product, 16% identity)(25, 39) .
Across all four proteins, the regions surrounding the endonuclease III
thymine glycol binding site (Ala-113 through Arg-119) and the putative
[4Fe-4S]
cluster motif
(C-X
-C-X
-C-X
-C)
were particularly well conserved. The nucleotide sequence for the M. luteus pdg gene has been submitted to GenBank and assigned
accession number U22181.
Purification scheme 3 accomplished two
goals: (i) a more streamlined purification procedure was developed, and
(ii) enough protein was isolated to characterize the enzyme. The 32-kDa
partner of the 31/32-kDa UV endonuclease pair was purified to apparent
homogeneity after three chromatographic steps: single-stranded
DNA-agarose, SP-Sepharose, and phenyl-Sepharose. Active 31-kDa protein
was eluted off the phenyl-Sepharose column prior to its 32-kDa partner,
but it was impure (data not shown). Pure 32-kDa protein was eluted off
the phenyl-Sepharose column in fractions 300-325 only after
extensive, postgradient washing with low salt buffer (Fig. 3A). Consequently, these fractions were very
dilute; each silver stain band represented 400 µl of
trichloroacetic acid-precipitated material. Each fraction was capable
of nicking a 49-mer oligonucleotide duplex containing a site-specific cis-syn cyclobutane thymine dimer. The resultant 20-mers
comigrated with the
-elimination product generated by T4
endonuclease V (Fig. 3B, lane 18). Fraction
330 lacked the 32-kDa protein and was incapable of nicking the 49-mer
substrate.
Figure 3:
Purification scheme 3: silver staining and cis-syn thymine dimer nicking activity across purified M.
luteus UV endonuclease fractions. Every fifth phenyl-Sepharose
fraction (400 µl/3 ml) across fractions 300-330 was
trichloroacetic acid precipitated and subjected to electrophoresis on a
15% SDS-PAGE gel. Panel A shows the silver stain of the gel.
The positions of five molecular size markers are noted to the left of the gel: ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa),
-lactamase (18.4 kDa), lysozyme (14.3 kDa), and bovine trypsin
inhibitor (6.2 kDa). The arrow to the right of the
gel points at UV endonuclease. The autoradiographic results of an
accompanying nicking assay are given in panel B. Enzyme (1.5
µl of each fraction, lanes 3-16), buffer (1.5
µl, lanes 1 and 2), or T4 endonuclease V (1.5
µl = 450 ng, lanes 17 and 18) was
incubated with control (odd lanes) or cis-syn thymine
dimer-containing (even lanes) duplex 49-mer (0.4 ng =
12.5 fmol). Reactions were allowed to proceed for 60 min at 37 °C
before they were terminated by the addition of formamide loading
buffer. Samples were then run on a 15% acrylamide gel containing 8 M urea. Lane M contained oligonucleotide sizing
markers ranging from 8 to 32 bases.
P-Labeled 49-mer
substrate and 20-mer product were visualized via autoradiography of the
wet gel.
Characterization of the M. luteus UV
Endonuclease
The recent synthesis by Smith and Taylor (33) of a set of deoxyoligonucleotide 49-mers containing
defined thymidylyl-(3`
5`)-thymidine photoproducts allowed us to
investigate the dimer specificity of purified UV endonuclease. The
32-kDa protein (phenyl-Sepharose fraction 315 from purification scheme
3) incised duplex 49-mer containing cis-syn but not trans-syn-I,), or Dewar thymine dimers (Fig. 4A). Nor could the UV endonuclease nick a duplex
37-mer containing a 5,6-dihydrouracil lesion, a substrate of
endonuclease III(40) , or duplex 49-mers containing either an
A:G or an A:C mismatch, known substrates of MutY (Fig. 4, B and C)(41) . Although it is not evident from the
exposure presented in Fig. 4C, MutY was able to cleave
the A:C substrate at 4% of the rate that it cleaved the A:G substrate.
UV endonuclease product PhosphorImager counts were slightly elevated
for the 5,6-dihydrouracil and A:G substrates relative to their
undamaged counterparts, but, at most, the counts approached 2% of the
positive control counts or <1% of total counts. Considering that
piperidine treatment was not required to detect the cleavage product of
the cis-syn thymine dimer-containing substrate and that more
UV endonuclease enzyme was introduced into the 5,6-dihydrouracil and
mismatch assays than into the pyrimidine dimer assays, its cis-syn pyrimidine dimer-specific nicking capability probably represents
the biologically relevant role of M. luteus UV endonuclease.
The purified protein nicked cis-syn dimer-containing DNA in a
concentration-dependent manner, and piperidine treatment did not
enhance the conversion of 49-mer to 20-mer product (Fig. 5).
Piperidine treatment did increase the mobility of the 20-mer reaction
product, however; removal of the 3` sugar fragment through
-elimination left a 3` phosphate terminus with a faster mobility
than the original aldehyde (data not shown). The fact that piperidine
treatment failed to convert a significant number of AP sites to
single-stranded breaks suggested that the AP lyase activity of the M. luteus UV endonuclease was at least as strong as its N-glycosylase activity. Had the UV endonuclease possessed a
weak AP lyase, AP sites would have remained in the DNA following the N-glycosylase cut, piperidine treatment would have created DNA
breaks, and an increase in 20-mer product would have been observed.
Since the dose dependence of the UV endonuclease was not perfectly
linear, i.e. 2.50 µl of enzyme converted 100% of the
49-mer to 20-mer, whereas 1.25 µl of the enzyme converted only 30%
of the substrate to product, the kinetics of the UV endonuclease were
examined (Fig. 6). As would be expected for a situation of
substrate excess or even for this situation of approximately equimolar
substrate and enzyme concentrations, initial reaction velocities were
proportional to the enzyme concentration: 1.25 µl, 1.02%
min
(r = 0.991); 2.50 µl, 1.87%
min
(r = 0.985); and 3.75 µl,
3.70% min
(r = 0.975).
Figure 4:
Substrate specificity of M. luteus UV endonuclease. Panel A, buffer (1.5 µl, odd
lanes) or phenyl-Sepharose fraction 315 (1.5 µl, even
lanes) was incubated with control (lanes 1 and 2), cis-syn (lanes 3 and 4), trans-syn-I (lanes 5 and 6),) (lanes 7 and 8), or Dewar (lanes 9 and 10)
thymine dimer-containing duplex 49-mer (0.4 ng = 12.5 fmol). Panel B, buffer (2.5 µl, lanes 1 and 4),
phenyl-Sepharose fraction 315 (2.5 µl, lanes 2 and 5), or endonuclease III (2 ng, lanes 3 and 6) was incubated with control (lanes 1-3) or
5,6-dihydrouracil-containing duplex 37-mer (0.8 ng = 32 fmol). Panel C, buffer (2.5 µl, lanes 1, 4, and 7), phenyl-Sepharose fraction 315 (2.5 µl, lanes
2, 5, and 8), or MutY (100 ng, lanes 3, 6, and 9) was incubated with A:T (lanes
1-3), A:G (lanes 4-6), or A:C (lanes
7-9) 50-mer duplex (0.5 ng = 16 fmol). All reactions
were allowed to proceed for 60 min at 37 °C before they were
terminated by the addition of formamide loading buffer (panel
A) or piperidine treatment (panels B and C).
Samples were then subjected to electrophoresis on a 15% polyacrylamide
gel containing 8 M urea. M lanes contained
oligonucleotide sizing markers ranging from 8 to 32 bases.
P-Labeled 49-mer substrate and 20-mer product were
visualized via autoradiography of the wet gels. To obtain quantitative
data, digital images of the autoradiographs were analyzed using
BioImage software.
Figure 5:
Concentration dependence of M. luteus UV endonuclease glycosylase and nicking activities on cis-syn thymine dimer-containing oligonucleotide duplex. Increasing
amounts of phenyl-Sepharose fraction 315 (2.5 µl and 2, 4, 8, 16,
32, 64, 128, 256, and 512
dilutions thereof) were incubated
with cis-syn thymine dimer-containing duplex 49-mer (0.4 ng
= 12.5 fmol) for 60 min at 37 °C. Reactions were terminated
either by placing the reaction tubes in a dry ice-EtOH bath (
) or
by heating the mixtures with 1 M piperidine for 30 min at 85
°C (
), a treatment that served to convert any remaining AP
sites to single-stranded breaks. After the addition of formamide
loading buffer, samples were subjected to electrophoresis on a 15%
polyacrylamide gel containing 8 M urea.
P-Labeled
49-mer substrate and 20-mer product were visualized by both
autoradiography and PhosphorImaging of the wet gel. The relative
amounts of substrate remaining and product generated were determined
using Image Quant software.
Figure 6:
Kinetics of M. luteus UV endonuclease nicking of cis-syn thymine dimer-containing
oligonucleotide duplex. Increasing amounts of phenyl-Sepharose fraction
315 (1.25 (
), 2.50 (
), or 3.75 (
) µl) were
incubated with cis-syn thymine dimer-containing duplex 49-mer
(0.4 ng = 12.5 fmol) for increasing lengths of time (0, 5, 10,
or 20 min) at 37 °C. Reactions were terminated by heating the
mixtures with 1 M piperidine for 30 min at 85 °C, a
treatment that served to convert any remaining AP sites to
single-stranded breaks. After the addition of formamide loading buffer,
samples were subjected to electrophoresis on a 15% polyacrylamide gel
containing 8 M urea.
P-Labeled 49-mer substrate
and 20-mer product were visualized by both autoradiography and
PhosphorImaging of the wet gel. The relative amounts of substrate
remaining and product generated were quantitated using Image Quant
software.
Evidence for an Imino Intermediate in the M. luteus
UV Endonuclease Reaction
The catalytic mechanism of the M. luteus UV endonuclease was hypothesized to proceed via an
imino intermediate like that of other N-glycosylase/AP lyases.
Such an intermediate was detected both indirectly by demonstrating that
cyanide inhibited the enzyme and directly by trapping the intermediate
as (a) covalent enzyme-substrate product(s) with the reducing agent
NaBH
. UV endonuclease was reacted with cis-syn dimer-containing duplex 49-mer in the presence of NaCl or
equimolar concentrations of NaCN (Fig. 7, A and B). Fifty percent inhibition of the reaction occurred around 6
mM NaCN, an IC
very similar to the 3-5
mM range observed for T4 endonuclease V(19) . Cyanide,
unlike NaBH
, reacts with an imino intermediate to form a
slowly reversible tetrahedral complex that cannot be isolated by
denaturing PAGE. Therefore, no shifted bands representing
enzyme-substrate complexes were seen in the Fig. 7A autoradiograph. Stable complexes were formed when NaBH
was present in the reaction at
10 mM (Fig. 7C). The complexes migrated more slowly than
the 49-mer substrate alone and were located just beneath the wells. It
is not understood how the upper and lower complexes differed, but they
were formed somewhat quantitatively according to the amount of enzyme
added to the reactions. The PhosphorImager covalent complex counts in Fig. 7C, lane 14, for instance, totalled
13,885, almost four times the 3,864 counts totalled in lane 7,
and translated into approximately 1% of the substrate molecules being
cross-linked to the enzyme. Fig. 7D illustrates
graphically that high salt concentrations whether of NaBH
or NaCl dramatically reduced the nicking activity of the M.
luteus UV endonuclease. A direct comparison of Fig. 7, B and D, would be misleading; it would lead one to
the conclusion that NaBH
inhibited the reaction much less
efficiently than NaCN. If a NaBH
inhibition experiment had
been conducted using 1 µl rather than 4 or 16 µl of UV
endonuclease, 50% inhibition probably would have occurred at a more
comparable level of NaBH
than the
30 mm concentration
observed in Fig. 7D.
Figure 7:
Inhibition by NaCN or NaBH
of M. luteus UV endonuclease nicking of cis-syn thymine
dimer-containing oligonucleotide duplex. Panel A, buffer (4.0
µl, lanes 1 and 2) or phenyl-Sepharose fraction
315 (4.0 µl, lanes 3-12) was incubated with control (lanes 1 and 3) or cis-syn thymine
dimer-containing (lanes 2 and 4-12) duplex
49-mer (0.8 ng = 25 fmol). NaCl (odd lanes) or NaCN (even lanes) was present in the reaction mixtures at 0 mM (lanes 1-4), 5 mM (lanes 7 and 8), 10 mM (lanes 9 and 10), or 25
mM (lanes 11 and 12). Panel C,
phenyl-Sepharose fraction 315 (4.0 or 16.0 µl, lanes 1-7 or 8-14) or buffer (16.0 µl, lane 15)
was incubated with cis-syn dimer-containing duplex 49-mer (0.8
ng = 25 fmol). NaCl or NaBH
was present in the
reaction mixtures at 0 mM (lanes 1, 8, and 15), 5 mM (lanes 2, 3, 9,
and 10), 10 mM (lanes 4, 5, 11, and 12), or 100 mM (lanes 6, 7, 13, and 14). All reactions were
terminated after 60 min at 37 °C by the addition of formamide
loading buffer, and the samples were subjected to electrophoresis on
15% polyacrylamide gels containing 8 M urea. M lanes contained oligonucleotide sizing markers ranging from 8 to 32
bases.
P-Labeled 49-mer substrate, 20-mer product, and
enzyme substrate complex(es) were visualized both by autoradiography
and PhosphorImaging of the wet gels. Panels A and C show the autoradiographic results of these experiments; panels
B and D summarize the PhosphorImager/Image Quant data.
The inset in panel C superimposes a 1-week exposure
of the gel over a 4-h autoradiograph. Although its placement is
essentially accurate, the inset was shifted downward just
slightly so as to not obscure the residues marking the wells. The inset arrows point at enzyme-substrate complexes. As is
indicated by the slash marks above the wells in panels A and C, the samples spread outward as they ran through the
polyurea denaturing gels; the residue that can be seen in each well
will not align perfectly with the 49-mer or 20-mer
bands.
DISCUSSION
For a number of years, our laboratory tried unsuccessfully to
clone the M. luteus UV endonuclease using strategies designed
to exploit the suspected homology between the M. luteus enzyme
and T4 endonuclease V(42) . As a final strategy, we chose to
purify the M. luteus UV endonuclease, sequence its amino
terminus, and design best guess oligonucleotide probes to screen a
randomly sheared M. luteus genomic
ZAP II library.
Contrary to what we had expected, the M. luteus UV
endonuclease turned out to be a 31/32-kDa protein, not a low molecular
weight protein in the range of 11,000-18,000. Furthermore, the
product of the M. luteus pdg gene resembled the E. coli repair proteins, endonuclease III and MutY, not endonuclease V.
The M. luteus UV endonuclease is intriguing in that it
constitutes the newest member of an emerging family of
[4Fe-4S]
cluster DNA repair glycosylases.
Moreover, these glycosylases possess widely divergent substrate
specificities. The M. luteus UV endonuclease cleaves DNA at
pyrimidine dimers, endonuclease III releases thymine glycols and a
number of other ring-saturated and ring-fragmented derivatives of
thymine(25) , and MutY removes undamaged adenines that are
mispaired with 8-oxoguanine lesions, 8-oxoadenine lesions, guanines, or
cytosines (41, 43) The function of the M.
thermoformicicum protein has not yet been determined, but it has
been speculated to be involved in the repair of G:T mismatches that
result from the deamination of 5-methylcytosine by another pFV1
plasmid-encoded protein, the GGCC-recognizing
methyltransferase(39) . Thus, the
[4Fe-4S]
cluster glycosylases either share
a common core structure onto which base-specific recognition motifs
have been added or recognize the distortion introduced by DNA damage as
opposed to the damage itself. At least in endonuclease III, the
[4Fe-4S]
cluster appears to contribute to
the structural integrity of the protein rather than to play a direct
role in catalysis(44, 45) . One additional piece of
evidence supports our hypothesis that the UV endonuclease and
endonuclease III are structurally similar: the SP-Sepharose Fast Flow
resin served as an invaluable tool in the purifications of both enzymes (46) . To eliminate any possibility that we had accidentally
cloned the M. luteus homolog of either endonuclease III or
MutY, we demonstrated that purified protein was incapable of cleaving a
5,6-dihydrouracil-containing substrate or mismatch substrates,
respectively.
Previous biochemical characterizations of the M.
luteus UV endonuclease have examined the enzyme's activity
on UV-irradiated DNA substrates that undoubtedly contained a mixture of
pyrimidine dimer types, cis-syn cyclobutane dimers being the
most prevalent followed by pyrimidine-pyrimidone) dimers. We examined
the dimer specificity of the UV endonuclease on four newly available
deoxyoligonucleotide 49-mers, each of which contained one the following
thymidylyl-(3`
5`)-thymidine photoproducts: a cis-syn, trans-syn-I,), or Dewar dimer(33) . Although the
enzyme nicked only at the cis-syn thymine dimer, literature
precedent did exist for this experiment; T4 endonuclease V can cleave a
site-specific 49-mer duplex containing a trans-syn-I dimer,
albeit at 1% of the rate that it cleaves 49-mer duplex containing a cis-syn dimer(33) . The major product of the M.
luteus UV endonuclease reaction comigrated with the T4
endonuclease V
-elimination product; and consistent with the
findings of Bailly et al.(21) , no
-elimination
was observed. We did not test whether or not
-elimination could be
forced under conditions of gross enzyme excess. Occasionally, a
secondary product was present between the conventional
- and
-elimination or piperidine product positions. It was never
ascertained whether this band was a relevant product or merely an
artifact of electrophoresis. The apparent specific activity of the
purified UV endonuclease was comparable to that of T4 endonuclease V,
but the M. luteus UV endonuclease possessed an AP lyase
activity equal to its N-glycosylase activity. Its propensity
not to dissociate prior to the
-elimination step may explain why
the M. luteus enzyme-substrate complex was trapped more
readily with NaBH
than the T4 endonuclease V imino
intermediate. Other investigators have reported that the N-glycosylase activity of the M. luteus UV
endonuclease is up to an order of magnitude greater than its apparent
AP lyase activity, even in partially purified preparations that may be
contaminated by multiple AP
endonucleases(2, 7, 8) . These findings are
difficult to reconcile with our own unless, as noted by Hamilton and
Lloyd(8) , the AP lyase activity is more labile over time than
is the N-glycosylase activity. Finally, high salt
concentrations (100 mM salt on top of 25 mM
NaH
PO
, pH 6.8 buffer) reduced the nicking
activity of the UV endonuclease. One of two explanations seems likely:
(i) either salt-sensitive processivity facilitated the cleavage of the
substrate even though it was only 49 bp long, or (ii) salt inhibited
the UV endonuclease via a mechanism that was independent of
processivity. The protein was clearly distinct from the M. luteus
35-kDa class II AP endonucleases A and B in that they require
Mg
and are sensitive to inactivation by
EDTA(47, 48) .
The catalytic mechanism of the M. luteus UV endonuclease has been shown to involve a Schiff
base intermediate. This finding strengthens our theory that all N-glycosylase/AP lyases proceed via an imino
intermediate(28, 29) . Lys-120, a basic residue, was
implicated recently in the reaction mechanism of endonuclease III;
mutagenesis of Lys-120 to Gln-120 resulted in a 10
-fold
decrease in the enzyme's activity compared with wild
type(25) . An analogous Lys-135 residue is present in the M. luteus enzyme. If and when a dependable expression system
for the M. luteus UV endonuclease has been developed, we will
actively investigate whether the
-amino group of Lys-135 is
responsible for the enzyme's catalytic activities. If necessary,
the same goal will be pursued using traditional biochemical techniques.
Finally, even though the M. luteus UV endonuclease may play
only a backup role in vivo to a uvrABC-like
nucleotide excision repair
system(49, 50, 51) , it may play an important
role in elucidating the structure/function relationships within the
family of [4Fe-4S]
cluster DNA repair N-glycosylases.