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INTRODUCTION |
In Escherichia coli, the proteins MutY, MutM, and MutT
are involved in defending against the mutagenic effects of
7,8-dihydro-8-oxo-guanine (8-oxoG or
GO)1 lesions, the most stable
products known due to oxidative damage to DNA (1, 2). The MutT protein
specifically eliminates 8-oxo-dGTP from the nucleotide pool (3-5). The
MutM protein (Fpg protein) removes both ring-opened purine lesions and
mutagenic GO adducts from DNA (6, 7). MutM removes GO lesions
efficiently from C/GO mispairs but poorly from A/GO mispairs (7). When C/GO is not repaired by MutM, adenines are frequently incorporated opposite GO bases during DNA replication (8, 9). A second round of
replication through this mismatch subsequently leads to a G·C to
T·A transversion (9-12). A role of the MutY pathway in E. coli is the removal of adenines misincorporated opposite GO or G
following DNA replication (1, 13, 14). This is consistent with the
phenotype of E. coli mutY (or micA) mutants, which have higher mutation rates for G·C to T·A transversions than
wild-type strains (15, 16). Similar repair pathways involved in the
defense against oxidized guanines are found in higher organisms. MutY
homologous activity was found in calf thymus and HeLa cell extracts
(17, 18). The amino acid sequence of human MutY homolog shares about
40% identity to E. coli MutY protein, but its encoded protein has not yet been characterized (19).
The short-patch MutY repair pathway specifically repairs A/GO and A/G
to C/GO and C·G, respectively, and corrects A/C to G·C at a much
lower rate (13, 16, 20-24). The MutY protein is a 39-kDa iron-sulfur
protein (24-26) with its N-terminal domain sharing structural
similarity with endonuclease III (endo III) and AlkA (24-28). This
includes the helix-hairpin-helix (HhH) and Gly/Pro ... Asp loop
motifs. Endonuclease III repairs thymine glycol and oxidized
pyrimidines in DNA (29, 30) and AlkA repairs methylated purines (31,
32). The C-terminal domain of MutY plays an important role in the
recognition of GO lesions (33), and AlkA has an additional N-terminal
domain of unknown function (31, 32).
DNA glycosylases in the endo III superfamily can be divided into two
groups (34, 35). Bifunctional DNA glycosylases, including endo III and
8-oxoG glycosylase, use the conserved lysine (Lys-120 in endo III and
Lys-249 in hOGG1) to form a Schiff base intermediate and also possess
strong AP lyase activity (27, 36-38). Monofunctional glycosylases such
as AlkA lack the conserved lysine and AP lyase activity (31, 32, 39).
Because MutY has a serine residue at this conserved position, it was
originally grouped as a monofunctional glycosylase (39). While several
groups failed to detect 3' apurinic/apyrimidinic (AP) lyase activity in
their MutY preparations (13, 22, 39-41), our laboratory and others
have reported that MutY has a weak AP lyase activity (14, 24, 33,
42-45). MutY can also cleave DNA containing an unmodified AP site
(43). Other supportive evidence for MutY AP lyase activity is that MutY
can form a covalent Schiff base intermediate with its DNA substrates
(33, 43, 44, 46, 47). Trapping the covalent protein-DNA complex with sodium borohydride has been used as a diagnostic tool for bifunctional glycosylase/AP lyases (35, 38, 39). Thus, MutY represents a unique
group of glycosylases because it does not have the conserved lysine but
can form a Schiff base intermediate with its DNA substrates. In this
paper, we have investigated the reaction mechanism of MutY.
We show that Asp-138 can act as a general base to activate a
nucleophile. The glycosylase and trapping activities were completely abolished in the D138N MutY protein but the DNA binding activity of
this mutant protein was not drastically different from that of the
wild-type enzyme. The N-terminal domains of MutY, residues 1-226 (M25)
(33) and residues 1-225 (p26) (43, 45) have been shown to retain
catalytic activity. We isolated the imino-covalent M25-DNA intermediate
and identified several lysine residues (Lys-142, Lys-157, and Lys-158)
of MutY in the proximity of the active site. Mutation of Lys-157 and
Lys-158 both individually and combined, had no effect on MutY
activities. Mutagenesis of Lys-142 to Ala confirmed that Lys-142 could
form a Schiff base with DNA. However, K142A MutY mutant still had
adenine glycosylase activity. Moreover, the K142A MutY mutant is
different from the wild-type enzyme by its possession of a
/
-elimination activity on DNA containing an AP/8-oxoG. Possible
reaction mechanisms of MutY are discussed.
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EXPERIMENTAL PROCEDURES |
Plasmids and Bacteria--
The entire mutY
gene in pJTW10-12 (24) was amplified by PCR with two primers:
Chang 222 (5'-GCGACGCATATGCAAGCGTCGCAATTTTC-3') and Chang 90 (5'-GCCGGAGGATCCCTAAACCGGCGCGCCAGTGC-3'). Similarly, the N-terminal
domain of the mutY gene corresponding to Met-1 to Gln-226
(M25) was PCR-amplified from pJTW10-12 by primers Chang 222 and
Chang 223 (5'-GCCGGAGGATCCCTACTGTTTCGGTTTTTTGCCCG-3'). The
primer Chang 222 contains an NdeI site at 5' ends of the
coding sequences, and primers Chang 90 and Chang 223 contain a
BamHI site after the stop codons. The PCR products were
purified from agarose gels, cut with NdeI and
BamHI, and cloned into an
NdeI/BamHI-digested pET11a expression vector. The
inserted MutY sequences in the resulting clones pMYW-1 for
the entire mutY gene and pJ16-146-13 for Met-1-Gln-226 were confirmed by DNA sequencing. The mutY gene and its
derivatives in the plasmid pET11a were under the control of the T7
promoter. The expression host of the mutY mutants, PR70 (Su-
lacZ X74 galU galK Smr
micA68::Tn10Kan obtained from M. S. Fox)
harboring the 
DE3 lysogen, was constructed according to the
procedures described by Invitrogen.
Site-directed Mutagenesis--
The QuickChange site-directed
mutagenesis kit from Stratagene was utilized to mutate Asp-138 of the
MutY gene to Asn. Two complementary oligonucleotides, Chang 224 (5'-GTAAGCACTTTCCGATTTTAAACGGTAACGTCAAAC-3') and Chang 225 (5'-GTTTGACGTTACCGTTTAAAATCGGAAAGTGCTTAC-3'), were used to
construct this mutation. Briefly, the oligonucleotides were
phosphorylated by polynucleotide kinase and used in a PCR reaction with
pMYW-1 (containing the wild-type mutY gene) as the template
according to the Expand High Fidelity PCR procedure (Roche Molecular
Biochemicals). The resulting nicked circular DNA was then purified and
digested with DpnI to remove the methylated and
hemi-methylated DNA. The digested DNA was then self-ligated and
transformed into XL-1 Blue cells (Stratagene). The mutant gene was
first screened for the generation of a DraI restriction site, then further confirmed by DNA sequencing.
The K158A mutant was constructed by the method of Kunkel et
al. (48). The 1.3-kilobase BamHI-SphI
fragment containing the mutY gene was excised from pMYW-1
and cloned into BamHI-SphI site of M13mp18 for
mutagenesis. Oligonucleotide Chang 259 (5'-CTGGCCTGGGAAAGCTGAGGTCGAGAAT-3') was used to construct the
mutation. The K158A mutant was first screened for the generation of an
AluI restriction site, then further confirmed by DNA sequencing.
Mutants K157Q, K157A-K158A double mutant, and K142A were constructed by
PCR splicing overlap extension method (49). Two complementary
oligonucleotides covering the mutation site were synthesized. The
oligonucleotide pairs containing the antisense sequence and Chang 222 as well as the oligonucleotide pairs containing the sense sequence and
Chang 90 were used as primers and pJTW10-12 was used as the template
to amplify the N-terminal and C-terminal regions of mutY,
respectively. Both purified PCR products were then mixed in a 1:1 ratio
and used as templates for another PCR reaction containing Chang 222 and
Chang 90 primers. The resulting PCR products were cloned into pET11a as
described above. All the oligonucleotides used are listed in Table
I. Mutants K157Q, K157A-K158A, and K142A
were first screened for the generation of MscI,
SalI, and XhoI restriction sites, respectively,
then confirmed by DNA sequencing.
Expression and Purification--
E. coli cells
PR70/DE3 harboring the expression plasmid containing MutY, MutY
mutants, or M25 were grown in LB broth containing 50 µg/ml ampicillin
at 30 °C. The expression of MutY and its derivatives were induced at
an A590 of 0.6 by the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 1 mM to the culture at 23 °C. The cells
were harvested 16 h later.
Mutant MutY proteins were quickly checked for activity in the crude
cell extracts from 50-ml cultures. Cells were centrifuged in a SS34
rotor at 10,000 rpm for 15 min. The cell pellets were resuspended in 4 ml of 20 mM potassium phosphate (pH 7.4), 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol and
sonicated for 6 cycles of 10 s, followed by 5 s of rest each
cycle. After centrifuging at 10,000 rpm in a SS34 rotor for 40 min at
4 °C, the supernatant was quickly frozen in small aliquots at
80 °C. Alternatively, cell extracts from 1 liter of E. coli cells were prepared as described previously (50).
M25 domain and MutY mutant proteins (D138N and K142A) were purified
from approximately 40 g of E. coli PR70/DE3 cells
harboring the respective overproduction plasmids, similar to the method used with wild-type enzyme (24). Nicking of A/G-containing 20-mer DNA
was assayed during the purification of the M25 domain. Binding of
A/GO-containing 20-mer DNA was assayed during the purification of the
MutY(D138N) and MutY(K142A) enzymes. As judged on a 12% SDS-polyacrylamide gel, all proteins were purified to >99%
homogeneity (data not shown). The purification of homogeneous MutY
protein from an overproducing E. coli JM109 strain,
harboring pJTW10-12, has been described previously (24).
Oligonucleotide Substrates and Enzymes--
Oligonucleotides of
19-mer containing base mismatches were labeled as described by Lu
et al. (14). E. coli uracil DNA glycosylase (UDG)
was purchased from Life Technologies, Inc. DNA substrates containing
U/G or U/GO (300 fmol) were fully converted to AP/G or AP/GO by
treating with 1.5 units of UDG at 37 °C for 1 h in MutY buffer
(20 mM Tris-HCl, pH 7.6, 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol).
MutY Binding, Cleavage, and Trapping Assay--
The MutY
activity assays with labeled oligonucleotide substrates were performed
as described by Lu et al. (44) with some modifications. MutY
enzyme was diluted with diluent (20 mM potassium phosphate,
pH 7.4, 50 mM KCl, 1.5 mM dithiothreitol, 0.1 mM EDTA, 200 µg/ml bovine serum albumin, and 50%
glycerol) before use. MutY binding and cleavage (glycosylase) buffer
contains 20 mM Tris-HCl, pH 7.6, 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9%
glycerol. MutY trapping buffer contains 20 mM Tris-HCl, pH
7.6, 1 mM dithiothreitol, 1 mM EDTA, and 2.9%
glycerol. For the glycosylase assays, unless specified in reactions,
samples after reactions were lyophilized to dry, resuspended in 3 µl
of formamide dye (90% formamide, 10 mM EDTA, 0.1% xylene
cyanol, and 0.1% bromphenol blue), heated at 90 °C for 2 min, and
loaded onto 14% 7 M urea sequencing gels.
Formation of Large Scale Enzyme-DNA Covalent Complex--
A
covalent complex of M25 with A/G-containing 19-mer DNA was formed in a
reaction containing 1.8 nmol of DNA and 1.8 nmol of M25 in 180 µl of
cleavage reaction buffer in the presence of 0.1 M
NaBH4. A NaBH4 stock solution was freshly
prepared and was added immediately after M25 was added. After
incubation at 37 °C for 30 min, 5× dye buffer (0.5 M
sucrose, 15% SDS, 312.5 mM Tris-HCl, pH 6.9, 10 mM EDTA, 5% -mercaptoethanol, and 0.0025% bromphenol
blue) was added to the samples, which were heated at 90 °C for 2 min
and separated on a 12% polyacrylamide gel in the presence of SDS
(SDS-PAGE) according to Laemmli (51). The SDS-polyacrylamide gel used
was cast 1-3 days before use with 0.1 M thioglycolate present in the running buffer to scavenge reactive compounds left in
the gel. Electrophoretic transfer to a PVDF membrane (Millipore) was
performed using a Trans-Blot cell. The membrane was pre-wet in 100%
methanol followed by transfer buffer (12.5 mM Tris-HCl, pH
8.3, 96 mM glycine, 10% methanol, and 0.001% SDS).
Proteins were transferred overnight at 4 °C at 30 V. The PVDF
membrane was washed and stained in 10% Ponceau S for 10 min and
destained in water.
Tryptic Digestion, HPLC Analysis, and N-terminal
Sequencing--
Both DNA-bound and free protein bands were excised
from the PVDF membrane and submitted to in situ digestion
with trypsin (52), substituting 0.1% Zwittergent 316 for Triton
RTX-100 in the digestion buffer. The resulting peptides were separated
by microbore high performance liquid chromatography using a Zorbax C18
1.0 mm by 150 mm reverse-phase column on a Hewlett-Packard 1090 HPLC/1040 diode array detector. Optimum fractions from the chromatograms were chosen based on differential UV absorbance at 205, 277, and 292 nm, and peak symmetry and resolution. Peaks were further
screened for length and homogeneity by matrix-assisted laser desorption
time-of-flight mass spectrometry on a Lasermat 2000 (Finnigan, Hemel,
United Kingdom). Selected fractions were subjected to automated Edman
degradation on a model 477A Sequencing System (Applied Biosystems,
Foster City, CA). Details of strategies for the selection of peptide
fractions and their microsequencing have been previously described
(53). Alternatively, tryptic peptide sequences were determined by
microcapillary HPLC/electrospray ionization/tandem mass spectrometry on
a Finnigan LCQ quadrupole ion trap mass spectrometer (San Jose, CA) as
described by Nash et al. (27).
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RESULTS |
Mutant MutY(D138N) Is Defective in Glycosylase Activity--
As
shown in Fig. 1, purified MutY(D138N)
mutant was inactive in the glycosylase assay with both A/G- and
A/GO-containing 20-mer DNA even at an enzyme concentration of 25 nM with an enzyme/DNA molar ratio of 278. However, in the
control reactions, wild-type MutY protein cleaved both A/G- and
A/GO-containing DNA. The trapping assay of MutY (D138N) in the presence
of sodium borohydride indicated that this mutant did not form any
covalent-complexes with A/G- and A/GO-containing DNA (data not shown).
Thus, these results confirmed that MutY(D138N) was defective in
glycosylase activity.
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Fig. 1.
Mutant MutY (D138N) is defective in
glycosylase activity. 1.8 fmol of 3'-end-labeled 20-mer
oligonucleotides containing A/G (panel A) and
A/GO (panel B) were incubated for 30 min at
37 °C with MutY (lane 1) or D138N mutant
protein (lanes 2-6) in 20 µl of reaction
buffer. The final enzyme concentrations were as follows: 0.5 nM (lane 1), 25 nM
(lane 2), 10 nM (lane 3), 5 nM (lane 4), 2.5 nM (lane 5), and 0.5 nM
(lane 6). Arrows indicate the
positions of intact oligonucleotide (I) and the nicked
product (N).
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Binding Affinity of the MutY(D138N) Mutant for Different
Mismatches--
In initial studies with crude extracts, we observed
that MutY(D138N) could bind A/G-and A/GO-containing DNA (data not
shown). Thus, the binding of A/GO-containing DNA was used to monitor
the activity during the enzyme purification. Further studies were then
performed to determine the dissociation constants of MutY(D138N) with
A/G- and A/GO-containing DNA substrates. DNA substrates were incubated
with various concentrations of MutY in binding assays, and experiments
were repeated at least three times. The apparent dissociation constants
(Kd) of MutY(D138N) and DNAs containing A/GO and
A/G mismatches were determined using a range of protein concentrations
with a fixed DNA concentration (Table
II). The apparent
Kd values of MutY(D138N) and DNAs containing
A/GO and A/G mismatches were 0.10 and 24 nM, respectively,
while the apparent Kd values of wild-type MutY
and A/GO- and A/G-containing DNAs were 0.066 and 5.3 nM,
respectively (14). The apparent Kd values of the
MutY(D138N) with A/GO- and A/G-containing DNA were 1.5- and 4.5-fold,
respectively, higher than that of the wild-type MutY protein. Despite
the fact that MutY(D138N) is defective in glycosylase activity, it is
capable of binding to both A/G- and A/GO-containing DNA substrates
although with slightly lower affinity than the wild-type.
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Table II
Apparent dissociation constants of MutY(D138N) and wild-type MutY
Binding constants for MutY are derived from Lu et al. (14).
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The binding of MutY(D138N) with DNA substrates containing other
mismatches was also tested. The apparent dissociation constants of the
mutant protein were compared with those of the wild-type MutY (Table
II). The binding affinities of MutY(D138N) with these substrates were
not significantly different from that of the wild-type MutY.
MutY(D138N) has 5-6-fold weaker binding to A/G-, Z/G-, A/2AP-, and
C/G-containing DNA (Z, 7-deaza-adenosine; 2AP, 2-aminopurine), but has
affinity to A/GO, A/C, A/I, N/G, and C/GO substrates (I, inosine; N,
nebularine) similar to that of wild-type MutY.
Determination of the MutY Residue Responsible for the Covalent
Complex Formation Detected by Peptide Sequencing--
The amino acid
sequences of E. coli MutY and endo III are highly homologous
(25, 26). However, although lysine 120 of E. coli endo III
has been suggested to be necessary for the formation of the
enzyme-substrate intermediate (37), MutY has a serine residue at this
position but can form Schiff base intermediate quite efficiently (33,
43, 44, 46, 47). To determine which amino acid residue in MutY is
involved in the nucleophilic attack upon the C1' carbon of the sugar of
adenosine, the covalent complexes of MutY with DNA and its
catalytically active domain (M25) with DNA were separated from the free
protein by SDS-PAGE and stained with Coomassie Blue. This experiment
was performed differently from other published results that used
labeled DNA and monitored the extent of bound substrates (33, 43, 44, 46, 47). As expected, Fig. 2 shows that
the protein-DNA complexes displayed slower mobility than the free
proteins. The molecular masses of the MutY-DNA and M25-DNA complexes
were estimated to be 45 and 31 kDa, respectively. About 25% of M25 and
MutY could be trapped with A/G-containing 20-mer DNA substrate at an
enzyme/DNA molar ratio of one in the presence of sodium borohydride.
The DNA-bound and free M25 from a large scale reaction were transferred to a PVDF membrane, digested with trypsin, and analyzed by HPLC. The
chromatograms (Fig. 3) showed that three
peptide peaks (peaks 35, 50, and 89) were reduced and four peptide
peaks (peaks 22-24, 25, 27, and 92) were increased in the DNA-bound
sample as compared with free protein. Edman degradation of peak 50 from
the free protein unfortunately revealed no sequence information. The
sequence of peak 35 of free M25 was determined to be HFILDGNYK
(residues 133-142). Based on the absorption at 204, 277, and 292 nm,
peaks 27 and 92 of the DNA-bound M25 sample were expected to be Trp- and Tyr-containing peptides, respectively. Peak 22/24 from DNA-bound M25 sample had unusual UV absorption and was determined to have the
sequence CYAVSGWPGK (residues 148-157) (Fig.
4). Mass spectrometric analysis of 22/24
M25B-PT identified a peak that corresponded to the size of residues
148-157 but not to residues 148-158. According to our initial model,
based on the endo III structure and the recently solved x-ray structure
of the MutY N-terminal domain with adenine bound in the active site,
-NH2 groups of these Lys residues are all located in the
proximity of the adenine active site pocket (28) (Fig.
5). Specifically, the -NH2
of Lys-142 is positioned in the same position as Lys-120 of endo III.
Although the data obtained are not definitive, they implicate that a
closely spaced Lys-142, Lys-157, or Lys-158 is a likely candidate to
provide the primary amine involved in forming a Schiff base with
DNA.
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Fig. 2.
Formation of covalent complexes of MutY and
M25 with A/G-containing DNA in the presence of NaBH4.
Oligonucleotide substrate (0.1 nmol) containing an A/G mismatch was
incubated with an equal molar amount of MutY (lane 2) or M25 (lane 3) in the presence of
0.1 M NaBH4 in 10 µl of trapping buffer at
37 °C for 30 min. Reactions were stopped by adding 5 µl of 3× dye
(9 mM Tris-HCl, pH 6.8, 15% glycerol, 3% SDS, 5%
-mercaptoethanol, and 0.3 mg/ml bromphenol blue) and the products,
after heating at 90 °C for 2 min, were electrophoresed on a 4-20%
gradient SDS-PAGE gel. The gel was stained with Coomassie Blue. The
positions of free proteins (F-M25 and F-MutY) and
covalent complexes (C-M25 and C-MutY) are
indicated. Molecular mass standards (New England Biolabs prestained
markers) were run in lane 1.
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Fig. 3.
HPLC chromatograms of trypsin-digested
products of free M25 (M25FR-PT, panel A) and M25-DNA complex (M25B-PT,
panel B). The DNA-bound and free M25
from a large scale reaction similar to Fig. 2 were transferred to a
PVDF membrane, digested with trypsin, and analyzed by HPLC as described
under "Experimental Procedures." UV absorbance was measured at 205 nm (top thin line with major peaks),
277 nm (middle thicker line), and 292 nm (bottom thin line with minor
peaks). The chromatograms showed that three peptide peaks (peaks 35, 50, and 89) (marked with stars in panel A and triangles in panel B)
were reduced and four peptide peaks (peaks 22-24, 25, 27, and 92)
(marked with inverted triangles in panel B) were increased in the DNA-bound sample as compared with
free protein.
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Fig. 4.
Some trypsin peptides of E. coli MutY. The residue numbers of lysines (K,
bold) and arginines (R, circled) were
indicated underneath. The sequences of peak 35 of M25FR-PT and peak
22/24 of M25B-PT were marked by boxes.
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Fig. 5.
The x-ray structure of MutY catalytic core
(28). The side chains of Asp-138, Lys-142, Lys-157, Lys-158, and
Ser-120 are shown in sticks and balls. Ser-120 is located in the
helix-hairpin-helix domain conserved in the endo III superfamily. The
iron-sulfur cluster located to the upper right of
the figure is represented as large spheres.
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Lys-142 of MutY Forms a Schiff Base with DNA--
Based on the
peptide sequencing of DNA-bound M25, residues Lys-142, Lys-157, or
Lys-158 could be involved in the Schiff base formation. Thus, K142A,
K157Q, K158A, and K157QK158A MutY mutants were constructed by
site-directed mutagenesis. These mutant proteins were expressed in
mutY mutant cells, and cell extracts were assayed for A/G
and A/GO glycosylase, trapping, and binding activities. The expression
of these mutant proteins was comparable to the wild type, except the
K157Q mutant protein was less soluble than the others (data not shown).
Mutation of Lys-157 and Lys-158 both individually and combined, had no
effect on the MutY activities. However, the K142A mutant protein was
unable to form the Schiff base intermediates with DNA substrates at an
enzyme/DNA molar ratio of 40 (Fig. 6). At
much higher enzyme/DNA molar ratios, a very small amount (<0.5%) of
covalent complex could be observed with the K142A mutant protein (data
not shown). Thus, -NH2 of Lys-142 is the amine used in
forming the Schiff base with DNA. However, all of these mutant proteins
retained their DNA binding activity (Fig.
7).
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Fig. 6.
Formation of covalent complexes of MutY
mutants with A/GO-containing DNA in the presence of
NaBH4. Oligonucleotide (3'-end-labeled 20-mer, 1.8 fmol) containing an A/GO mismatch was incubated with MutY or cell
extracts containing expressed MutY mutant proteins in the presence of
NaBH4. Reactions were carried out in 20 µl of trapping
buffer at 37 °C for 30 min. Lanes 1-6 used
100 ng of cell extracts prepared as described (50): lane 1, K142A; lane 2, wild-type MutY;
lane 3, vector pET11a; lane 4, K157Q; lane 5, K158A;
lane 6, K157A/K158A double mutant.
Lane 7 contained 3.6 nM purified
MutY. The products after heating at 90 °C for 2 min were
electrophoresed on a 12% SDS-PAGE gel. The gel was dried and
autoradiographed. The positions of free DNA (F) and covalent
complex (C) are indicated.
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Fig. 7.
Binding of the A/GO-containing
oligonucleotide by MutY mutants. A/GO-containing 20-mer
oligonucleotide was assayed for binding with 3.6 nM
purified MutY (lane 2) or extracts of MutY
mutants (lanes 3-14) at 37 °C for 30 min.
Lane 1 represents DNA alone. Lanes
labeled with H used 100 ng and lanes labeled with
L used 20 ng of cell extracts prepared as described (50).
Lanes 3 and 4, vector pET11a;
lanes 5 and 6, wild-type MutY;
lanes 7 and 8, K142A; lanes 9 and 10, K157Q; lanes 11 and 12, K158A; and lanes 13 and
14, K157A/K158A double mutant. The products were analyzed on
an 8% native gel. Arrows indicate the positions of MutY-DNA
complex (B) and free DNA (F).
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K142A Mutant MutY Has DNA Glycosylase Activity--
To test
whether K142A retained the glycosylase activity of native MutY, three
reactions were carried out for the cleavage assays. In the first
reaction, the samples were directly loaded onto the sequencing gel
without heating, samples in the second reaction were heated at 90 °C
for 2 min before loading to the gel, and samples in the third reaction
were treated with 1 M piperidine for 30 min (this condition
promotes -elimination) after the MutY reaction as well as being
heated at 90 °C for 2 min before loading onto the gel. As shown in
Fig. 8, both MutY and K142A mutant could cleave 3'-labeled A/G-containing DNA. It appeared that the K142A mutant
was even more active than the wild-type MutY. Both the K142A mutant and
the wild-type MutY had similar kinetics of glycosylase activity in
time-course studies (data not shown). Heating the samples at 90 °C
for 2 min (lanes 3 and 6) enhanced the
cleavage activities. However, further treatment of the products with
piperidine at 90 °C (lanes 4 and 7)
did not significantly increase the extent of cleavage by both MutY and
K142A proteins. Thus, both the K142A mutant and wild-type MutY possess
the adenine glycosylase activity.
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Fig. 8.
Glycosylase activities of wild-type and K142A
MutY. 1.8 fmol of 3'-end labeled A/G-containing 20-mer
oligonucleotide was incubated with 7.2 nM MutY
(Y lanes) or K142A mutant (A lanes) in 10 µl of glycosylase buffer at 37 °C for 30 min. Lane 1 represents untreated DNA. In reaction
I, samples were dried down, resuspended in formamide dye, and loaded
onto the sequencing gel. In reaction II, samples were resuspended in
formamide dye, heated at 90 °C for 2 min, and then loaded to the
sequencing gel. In reaction III, after enzyme reaction, piperidine was
added to the samples to a final concentration of 1 M and
samples were heated at 90 °C for 30 min and treated as reaction II.
DNA samples were fractionated on a 14% sequencing gel, and the gel
autoradiographed. Arrows indicate the positions of intact
oligonucleotide (I) and nicking product
(N).
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To address whether the products in the heated lanes are derived by
heat-promoted -elimination or AP lyase activity, we analyzed the
reaction products of monofunctional UDG (36). The AP/GO-DNA generated
by UDG was heated at 90 °C for 2 min at two salt concentrations. At
a low salt concentration (Fig. 9,
lane 5), no significant cleavage was observed. At
a high salt concentration, a condition used in the MutY assays, about
15% of the AP site was cleaved (Fig. 9, lane 8)
as compared with the sample treated with piperidine at 90 °C (Fig.
9, lane 6). At a high enzyme to DNA ratio, more
than 60% of A/G-containing DNA can be cleaved with heat treatment but without piperidine treatment (data not shown) (14, 44). Although heating promoted -elimination, it did not contribute to all the cleavage observed in the MutY reaction. It appears that heat is required to release the MutY protein from the cleaved products, as some
retarded and smeared DNA, migrating slower than the intact DNA, was
detected in non-heated samples (data not shown).
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Fig. 9.
Activity of MutY and K142A mutant proteins
with AP-containing DNA. Lanes 1-3 are 1.8 fmol of 5'-labeled DNA substrates containing U/GO. Lanes 4-14 are 1.8 fmol of 5'-labeled DNA substrates containing
AP/GO. AP/GO-DNA was obtained by treating 300 fmol of labeled
U/GO-containing DNA with 1.5 units of UDG in MutY cleavage buffer.
Samples (1 µl) in lanes 1-6 were supplemented
with 1 µl each of water and formamide dye, and samples in
lanes 7 and 8 were supplemented with 1 µl each of 10× MutY buffer and formamide dye. Samples in
lanes 2, 5, and 8 were
heated at 90 °C for 2 min (II) before loading to the
sequencing gel. Samples in lanes 3 and
6 were treated with 0.1 M piperidine at 90 °C
for 30 min (III). 3.6 fmol of 5'-end labeled
AP/GO-containing 20-mer oligonucleotides were incubated with MutY
dilution buffer (d, lanes 9 and
10), 14.4 nM MutY (Y,
lanes 11 and 12), or K142A mutant
protein (A, lanes 13 and
14) in 10 µl of reaction buffer at 37 °C for 30 min.
Samples were supplemented with 5 µl of formamide dye, divided into
two tubes (one heated (II) and one without heat
(I)). DNA samples were fractionated on a 14% sequencing gel
at 1200 V. The arrows mark the positions of intact
oligonucleotide (I), products with a 3'- ,-unsaturated
aldehyde via a -elimination (B), and products with a
3'-phosphate via /-elimination by piperidine
(P).
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K142A Mutant MutY Can Cleave AP-containing DNA--
If MutY truly
possesses AP lyase activity, it should be able to cleave a
phosphodiester bond at an AP site. 5'-Labeled AP/G-containing DNA was
reacted with K142A and MutY. To minimize heat-promoted -elimination,
after cleavage reactions, samples were kept in a low salt concentration
and one third of the formamide dye. The sequencing gel was run at 1200 V at 32 °C. As compared with the dilution buffer alone (Fig. 9,
lanes 9 and 10), MutY did contain a
weak AP lyase activity (Fig. 9, lanes 11 and
12). Quantitation of the PhosphorImager images indicated
about 5-10% of AP-DNA was cleaved by MutY after background
subtraction. The MutY AP lyase activity was consistently observed
higher than the background and increased when the enzyme concentration
increased (data not shown). The AP lyase activity is about 20% of that
of MutY glycosylase (compare lanes 3 and
4 of Fig. 8 to lanes 11 and
12 of Fig. 9).
When AP/GO-DNA was cleaved with the K142A protein, there was a new band
(marked with P in Fig. 9, lanes 13 and
14) migrating to the same position as that in the
piperidine-treated sample (Fig. 9, lane 6). This
product with a 3'-phosphate appeared even without heating at 90 °C
for 2 min. Thus, the K142A mutant MutY is different from the wild-type
enzyme by its possession of a /-elimination activity on DNA
containing an AP/GO. This surprising result supports the idea that the
K142A mutant MutY protein incises both 3' and 5' of an AP site,
although it does not form the Schiff base intermediate (Fig.
10).
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Fig. 10.
Possible reaction mechanisms of MutY.
A, reactions of wild-type MutY. Step 1, Asp-138 activates a water molecule, which then attacks
the C1' carbon to release the adenine base; step 2, the deoxyribose ring is opened; step 3, Lys-142 is promoted by a general base to attack the C1'
of the opened sugar. Schiff base is formed by several chemical
conversions. Step 1', Asp-138 deprotonates the
-amine of Lys-142. This nitrogen nucleophile displaces the
mismatched adenine from the DNA. Step 2', the
Schiff base tautomerizes to the ring-opened form. Step 4, the Schiff base undergoes -elimination and leads to
the strand cleavage. The products are a fragment with a
3'-,-unsaturated aldehyde and a fragment with a 5'-phosphate
group. Step 5, treatment with sodium borohydride
reduces the Schiff base to a stable covalently linked enzyme-DNA
complex. B, reactions of K142A mutant protein.
Steps 1 and 2 are similar to
steps 1 and 2 of A;
step 3, the DNA with an abasic deoxyribose is
cleaved by K142A mutant MutY through -elimination directly without
the Schiff base formation; step 4, K142A can
carry out -elimination and release the unsaturated sugar. Base
(piperidine or NaOH) treatment can also cause -elimination. The
resulting DNA has a 3'-phosphate end.
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Binding Affinity of the MutY(K142A) Mutant for DNA
Substrates--
Because the K142A mutant protein cleaves DNA using a
different reaction mechanism from that of the wild-type enzyme, binding affinities of these two proteins were compared by gel retardation. The
apparent dissociation constants (Kd) of purified
K142A protein with A/G- and A/GO-containing 20-mer DNA were 10.2 ± 2.6 and 0.057 ± 0.011 nM, respectively. The
MutY(K142A) has 2-fold lower affinity with A/G-containing DNA than the
wild-type but has affinity with A/GO substrate similar to that of
wild-type MutY (Table II) (14).
The binding of MutY and the K142A mutant with AP/GO-containing DNA was
also tested because the AP-DNA intermediate can form a Schiff base with
the wild-type enzyme but is excised by the K142A mutant through
/-elimination without Schiff base formation (Fig. 10).
Interestingly, the dissociation constant of the K142A mutant protein
with AP/GO-containing 20-mer DNA is 1.03 ± 0.26 nM,
which is 6-fold higher than that of MutY (Kd is
0.18 ± 0.11 nM).
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DISCUSSION |
Does MutY Have AP Lyase Activity?--
MutY has been classified as
both a monofunctional and a bifunctional enzyme. While several groups
failed to detect AP lyase activity in their MutY preparations (13, 22,
39-41), our laboratory and others have reported that MutY has a weak
AP lyase activity (14, 24, 33, 42-45). The covalent complex formation
of MutY or M25 with A/G-containing DNA is quite efficient (Fig. 2).
About 25% (sometimes up to 50%) of enzyme can be trapped with DNA
substrates at an enzyme/DNA molar ratio of 1 in the presence of sodium
borohydride. MutY can also be cross-linked to DNA substrates containing
an unmodified AP site but with less efficiency
(47).2 The Schiff base
formation is characteristic of bifunctional glycosylase/AP lyases (35,
38, 39). We also confirmed that MutY could cleave AP-DNA with a weak AP
lyase activity and that this AP lyase activity was about 20% of the
MutY glycosylase activity. Taken together with our previous results
that MutY can cleave more than 60% of A/G-containing DNA without
piperidine treatment (14, 44), we propose that MutY is not a
monofunctional glycosylase. Our results are slightly different from
that of Manuel and Lloyd (43), who reported that AP lyase activity was
as active as MutY glycosylase activity. The discrepancy may be caused
from heating the DNA samples before loading and/or high temperature of
the sequencing gel run at a high voltage.
Zharkov and Grollman (47) attributed the AP lyase activity of MutY,
observed, as an artifact of heating at 90 °C in Tris-containing buffer and thus concluded that MutY did not cleave the phosphodiester bond. However, a small amount of cleavage was observed in reactions without the heating step. We have reported that MutY nicking product could be detected in a native gel in the binding assays (14) in which
samples were never heated above 37 °C. To address whether heat can
promote -elimination, the AP/GO-DNA generated by UDG was heated at
90 °C for 2 min at two different salt concentrations. At a low salt
concentration, no significant cleavage was observed, but, at a high
salt concentration, a condition used in our MutY assays, about 15% of
the AP site was cleaved. Although heating promoted -elimination, it
did not contribute to all the cleavage observed in the MutY reaction
because the cleavage product of AP-DNA by MutY could be detected even
without heat.
Williams and David (46) compared the DNA cleavage between MutY and UDG
and suggested that MutY is a monofunctional glycosylase. Their assays
included a heating step, but limited cleavage was observed with 30-mer
substrates and some cleavage was observed with 18-mer DNA in the
reactions without NaOH treatment. Zharkov and Grollman (47) compared
MutY proteins from three laboratories including ours and found they
behaved similarly. Therefore, the conflict reported in the literature
for the MutY properties may be due to the length and sequence contents
of the DNA substrates as well as reaction conditions used in different laboratories.
Asp-138 Is a Catalytic Residue--
Asp-138 residue of MutY is
highly conserved in the endo III superfamily and has been implicated at
the active site to activate a nucleophile (27, 28, 32, 38).
Site-directed mutagenesis of Asp-138 to Gln in MutY totally ablated its
DNA glycosylase and trapping activities. Thus, these results confirmed
that Asp-138 is involved in MutY glycosylase activity. Based on the
different binding affinities between the D138N mutant and wild-type
MutY, we propose that Asp-138 may play another role in DNA substrate binding.
Lys-142 Forms the Protein-DNA Covalent Complex--
A central
issue in DNA glycosylase/AP lyase action is the identity of the enzyme
amine that forms the Schiff base intermediate. Two approaches were
taken to identify the active site amine of MutY. One approach is to
analyze the imino protein-DNA complex and determine the DNA-bound amino
acid by peptide sequencing. The other approach involves site-directed
mutagenesis which confirms that Lys-142 can form a Schiff base with
DNA. Inspection of the structure of the MutY catalytic domain and of a
model for how this domain might bind to DNA (28) suggests that the
-amine of Lys-142 is close to the catalytic Asp-138 (Fig. 5). Our
result is in agreement with this prediction and the data of Zharkov and Grollman (47), who also showed Lys-142 of MutY is involved in cross-linking to DNA.
The HPLC chromatograms in Fig. 3 show that three peptide peaks (peaks
35, 50, and 89) were reduced and four peptide peaks (peaks 22-24, 25, 27, and 92) were increased in the DNA-bound sample as compared with
free protein. We have identified the peak 35 containing Lys-142, but
unfortunately, the DNA-bound polypeptide was not identified.
Originally, peak 22/24 from DNA-bound M25 sample was suspected to bind
DNA but it turned out to be a polypeptide (Cys-148-Lys-157) close to
Lys-142 (Fig. 4). Thus, Lys-157 appears to be more susceptible to
trypsin digestion in the DNA-bound enzyme. This suggests that there are
some conformation changes upon substrate binding. With respect to MutY
structure, the helix containing Lys-157 and Lys-158 is positioned next
to Lys-142 and Asp-138 (Fig. 5); thus, a conformational change is likely.
Model for MutY Action--
Based on our results, we propose two
possible reaction mechanisms for the wild-type MutY. In one model,
Asp-138 activates a water molecule to attack the C1' carbon (Fig.
10A, step 1) (35). The adenine base is
released by this DNA glycosylase activity. The resulting deoxyribose
undergoes tautomerization with equilibration favoring the closed ring
structure (Fig. 10A, step 2). Lys-142 of MutY is promoted by a general base to attack the C1' of the opened
sugar. This second nucleophilic attack generates a trappable Schiff
base by several chemical conversions (Fig. 10A,
step 3). Alternatively, the original nucleophilic
attack at C1' involves the -amine of Lys-142. Asp-138 deprotonates
the -amine of Lys-142. This nitrogen nucleophile displaces the
mismatched adenine from the DNA leading to a Schiff base formation
(Fig. 10A, steps 1'-2'). The Schiff
base intermediate of MutY-DNA is quite stable (47). Wild-type MutY can
promote weak -elimination thus strand cleavage occurs through step 4 (Fig. 10A). The products are two fragments: one with a
3'-, -unsaturated aldehyde and one with a 5' phosphate group.
Lys-142 Has a /-Elimination Activity--
The most
surprising results from this study are that the MutY K142A mutant still
has adenine glycosylase activity on A/G- and A/8-oxoG-containing DNA
and that this mutant gains a new -elimination activity not possessed
by wild-type MutY. These properties are different from other
bifunctional glycosylase/AP lyases in which mutation of the conserved
lysine abolishes catalysis (27, 37, 54). Since K142A has glycosylase
activity, the mutant protein must use a water molecule to attack the
C1' of the adenosine (Fig. 10B, step
1). When Lys-142 is mutated to Ala, it is possible that a
water molecule occupies the position of -amine of Lys-142.
The K142A mutant MutY is different from the wild-type enzyme by its
possession of a /-elimination activity toward DNA containing an
AP/8-oxoG mismatch. In step 3 (Fig. 10B), DNA with an abasic deoxyribose can be cleaved by K142A MutY through -elimination directly without the Schiff base formation. MutY(K142A) then carries out a -elimination and subsequently releases the unsaturated sugar
(Fig. 10B, step 4). The final products
are a DNA fragment with a 3'-phosphate end, a fragment with a 5'
phosphate group, and an unsaturated sugar. In this regard, the K142A
MutY mutant behaves like the Fpg (MutM) protein, incising both 3' and
5' to the lesion (2, 55-57) but the K142A MutY does not form the
Schiff base intermediate. The mechanism involved in the
/-elimination by MutY(K142A) requires further investigation.
The mechanistic switch between K142A and wild-type MutY raises an
interesting question about the mechanistic preference of MutY since the
biological significance of the Schiff base formation of wild-type MutY
is not clear. Because MutY turns over extremely slowly, especially with
A/GO substrates (14, 34, 58), the -amine of Lys-142 may just happen
to lie closely to the active site and experience a chance encounter
with the AP site (Fig. 10A, step 3).
The reaction may be similar to the reported lyase activity of small
lysine-containing peptides on abasic sites in DNA (59). Alternatively,
the Schiff base formation may play an active role (Fig. 10A,
steps 3 and 2'). It has been shown
that the MutY-DNA covalent complex has a long half-life (47). MutY binding to its substrate in a stable covalent form may prevent Fpg from
reacting with the AP/GO-DNA or may recruit other proteins for repair
and re-synthesis. In the case of the second possibility, one would
expect the K142A mutant protein to have a weaker affinity to AP-DNA
than the wild-type enzyme. Our data seem to favor this notion because
the dissociation constant of K142A with AP/GO-containing DNA is 6-fold
higher than that of the wild-type MutY.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 410-706-4356;
Fax: 410-706-1787; E-mail: aluchang@umaryland.edu.
