Carcinogenesis vol.20 no.1 pp.103–108, 1999
Expression of the inactive C145A mutant human O6-alkylguanineDNA alkyltransferase in E.coli increases cell killing and mutations
by N-methyl-N9-nitro-N-nitrosoguanidine
Suvarchala Edara, Sreenivas Kanugula and
Anthony E.Pegg1
Department of Cellular and Molecular Physiology, Milton S. Hershey
Medical Center, Pennsylvania State University College of Medicine,
Hershey, PA 17033, USA
1To
whom correspondence should be addressed
Email: [email protected]
Human O6-alkylguanine-DNA alkyltransferase (AGT)
counteracts the mutagenic and toxic effects of methylating
agents such as N-methyl-N9-nitro-N-nitrosoguanidine
(MNNG) by removing the methyl group from O6-methylguanine lesions in DNA. The methyl group is transferred
to a cysteine acceptor residue in the AGT protein, which
is located at residue 145. The C145A mutant of AGT in
which this cysteine is converted to an alanine residue is
therefore inactive. When this C145A mutant was expressed
in an Escherichia coli strain lacking endogenous alkyltransferase activity, the number of G:C→A:T mutations actually
increased and the toxicity of the MNNG treatment was
enhanced. These effects were not seen when an E.coli strain
also lacking nucleotide excision repair (NER) was used.
The enhancement of mutagenesis and toxicity of MNNG
produced by the C145A mutant AGT was not seen with
another inactive mutant Y114E that contains a mutation
preventing DNA binding, and the double mutant C145A/
Y114E was also ineffective. These results suggest that the
C145A mutant AGT binds to O6-methylguanine lesions in
DNA and prevents their repair by NER. The inactive C145A
mutant AGT also increased the number of A:T→G:C
transition mutations in MNNG-treated cells. These
mutations are likely to arise from the minor methylation
product, O4-methylthymine. However, expression of wildtype AGT also increased the incidence of these mutations.
These results support the hypothesis that mammalian AGTs
bind to O4-methylthymine but repair the lesion so slowly
that they effectively shield it from more efficient repair
by NER.
Introduction
A major part of the toxicity of methylating agents is due to
the formation of O6-methylguanine in DNA (1–3). This adduct
causes the guanine to be copied incorrectly by DNA polymerase
inserting a thymine residue and thus producing G:C→A:T
mutations (4,5). O6-methylguanine in DNA can also cause cell
death, which occurs by apoptosis (6–9). Such killing is
mediated via the mis-match repair system (10,11). This system
recognizes the O6-methylguanine:thymine/cytosine pair as a
mis-match and causes a section of DNA strand containing the
thymine/cytosine to be degraded. When this section is filled
Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; IPTG, isopropyl
β-D-thiogalactopyranoside; MNNG, N-methyl-N9-nitro-N-nitrosoguanidine;
NER, nucleotide excision repair.
© Oxford University Press
in by DNA polymerase, the same error is re-inserted and a
futile cycle of DNA synthesis and degradation that leads to
cell death is set up. Although most of the work in which the
toxic effects of methylating agents has been studied was
carried out with compounds such as N-methyl-N9-nitro-Nnitrosoguanidine (MNNG) and N-methyl-N-nitrosourea, these
agents generate a similar methylating species to therapeutic
methylating agents such as procarbazine, dacarbazine and
temozolomide. Repair of O6-methylguanine may be a major
factor in resistance to these drugs but is also likely to reduce
their genotoxicity that leads to the incidence of secondary
tumors (12–14).
O6-alkylguanine-DNA alkyltransferases are a ubiquitous
family of DNA repair proteins that play an important role in
counteracting the toxic effects of methylating agents (15–18).
They act by transferring the methyl group from the O6-position
of guanine to a cysteine acceptor residue in the human
O6-alkylguanine-DNA alkyltransferase (AGT) protein. This
restores the DNA structure in a single step and very efficiently
protects against mutagenesis and killing by methylating agents
provided that adequate AGT is available. AGT, the human
member of this family, contains 207 amino acids and has the
acceptor site located at cysteine 145 (17,18).
The reaction between AGT and its substrate is thought to
be brought about by the generation of a thiolate anion at a
cysteine located in the active site of the protein. This facilitates
transfer of the alkyl group in an SN2 reaction from the DNA
to the cysteine acceptor in the AGT sequence leading to the
formation of S-methylcysteine (12,13,19). This reaction is
irreversible and each molecule of AGT can only be used once,
as the S-methylcysteine is not converted back to cysteine.
As expected for a DNA repair protein, alkyltransferase binds
to DNA although the mechanism of binding is not well
understood and two different models have been put forward
(20,21). Both of these are based on the crystal structure for
the Ada-C alkyltransferase from Escherichia coli (20,22). This
structure was of the protein alone and did not contain a bound
substrate. The cysteine acceptor site is not readily accessible
in this structure suggesting that a change in the protein
conformation upon binding DNA is necessary in order to
permit access of the target O6-alkylguanine. Conformational
changes in the protein as a result of binding to DNA have
been detected by using CD and fluorescent anisotropy (23–
25). Alterations in DNA conformation after AGT binding
consistent with local melting have been detected by near-UV
CD spectral changes (26). Other studies of the repair of doublestranded oligodeoxyribonucleotides containing O6-methylguanine analogs suggested that the duplex has to open up in
order for the reaction to take place (27). These results are
consistent with a base flipping mechanism in which the O6alkylguanine is flipped out of the DNA helix to permit the
reaction to occur. Such base flipping is now well established
for DNA repair enzymes and has been suggested for AGT
(21,28–31).
103
S.Edara, S.Kanugula and A.E.Pegg
Fig. 2. Expression of mutant AGT proteins in GWR109 cells. Extracts from
GWR109 cells expressing human wild-type AGT (lanes 1 and 2) or its
mutants C145A (lanes 3 and 4) and Y114E/C145A lanes 5 and 6) and
control GWR109 cells (lanes 7 and 8 ) were resolved by SDS–PAGE,
transferred to nitrocellulose and developed using antibodies to a peptide
corresponding to amino acids 8–20 of the human AGT. In lanes 1, 3, 5 and
7, 5 µg of protein were loaded and in lanes 2, 4, 6 and 8, 10 µg of protein
were used.
isolation kit was purchased from BIO101 (Vista, CA). DH5α MCR cells were
purchased from Bethesda Research Laboratories (Gaithersburg, MD). All
oligodeoxynucleotides were made in the Macromolecular Core Facility,
Hershey Medical Center, using a Milligen 7500 DNA synthesizer.
Fig. 1. Effect of MNNG on mutations and killing in GWR109 cells
expressing AGT mutant C145A. Results are shown for mutations to
revertants to ability to grow without histidine (a) and for survival (b) in
cells expressing no AGT protein (closed triangles) or wild-type AGT
(closed inverted triangles), C145A mutant AGT (open circles), Y114E
mutant AGT (closed circles), SAMDC (closed squares) and the double
mutant Y114E/C145A AGT (open triangles) were treated with different
concentrations of MNNG as shown. The values that were statistically
different (P , 0.01) from the SAMDC used as a control are marked with
asterisks. Results are means 6 SD for at least four estimations.
AGT binding to DNA with and without O6-methylguanine
adducts has been detected by gel shift experiments, footprinting
(32) and sedimentation analysis (33). In such experiments,
when oligodeoxynucleotides containing O6-methylguanine are
used, it is very hard to prevent the reaction of wild-type AGT
with the substrate. Therefore, the C145A mutant AGT has
been used as a surrogate (32). This mutant in which the normal
methyl acceptor site is converted to an alanine has been shown
to have no DNA repair activity in in vitro assays (34,35).
In the course of studies on the ability of wild-type AGT
and mutants to protect cells from the toxic effects of alkylating
agents in which C145A AGT was used as a negative control,
we have now observed that this C145A mutant AGT actually
increases the toxic effects of MNNG. The present paper gives
a detailed description of this phenomenon and discusses the
underlying biochemical mechanism responsible for it.
Materials and methods
Materials
All restriction enzymes were purchased from New England Biolabs (Beverly,
MA) and Gibco BRL (Gaithersburg, MD). Isopropyl β-D-thiogalactopyranoside
(IPTG), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranose and phenyl-β-D-galactoside, MNNG, ampicillin, kanamycin, chloramphenicol and all electrophoresis and media reagents were purchased from Sigma (St Louis, MO). Taq I
DNA polymerase was purchased from Amersham (Arlington Heights, IL).
Nitrocellulose was purchased from Schleicher and Schuell (Keene, NH).
Tween-20 was purchased from Bio-Rad (Hercules, CA). GENECLEAN DNA
104
Bacterial strains
The bacterial strains GWR109 (36), FC218, FC326 (37) and CJM2 (38) were
a generous gift from Dr L.Samson (Department of Molecular and Cellular
Toxicology, Harvard School of Public Health, Boston, MA).
Plasmids
All AGT mutants were expressed in E.coli using the pINIII-A3(lppP–5)
expression vector (39). Preparation of this vector plasmid containing sequences
for expression of the E11Q mutant of S-adenosylmethionine decarboxylase
(40), wild-type human AGT (41), C145A mutant AGT (35) and Y114E mutant
AGT (42) have been described. The pIN plasmid containing the Y114E/
C145A double mutant was made by three rounds of PCR with conditions as
described (43). The first round was carried out using pINAGT as template
and primers 59-CCCATCCTCATCCCGGCCCACAGAGTGGTCTGC-39
(mismatch underlined) to introduce the C145A mutation and primer A (59TTTAGCAGCCTGAACGTCGG-39) matching a nucleotide sequence downstream to the AGT stop codon. Second-round PCR was then carried out using
pIN-Y114E plasmid DNA as template using a primer B (59-CAGCTATGACCATGATTACGGATTC-39) matching a nucleotide sequence upstream to AGT
start codon and the 265 bp first-round PCR product excised from 1% agarose
gels. The product from the second-round PCR was then used as a template
in the third round PCR using primers A and B. The third-round PCR product
was ethanol precipitated, digested with EcoRI and BamHI enzymes and ligated
into pIN vector digested with the same enzymes to form pIN-Y114E/C145A.
The entire AGT protein coding sequence of this plasmid was verified by
sequencing and no additional mutations were found. The plasmids were
transferred into the bacterial strains by electroporation.
Determination of AGT protein expression
The amount of recombinant human AGT or its mutants expressed in GWR109
cells was determined by western blotting after separation by SDS–PAGE on
16% gels as described (44) using antibody MAP-1, which was raised to
peptide KRTTLDSPLGKLE corresponding to residues 8–20 of the human
AGT amino acid sequence (45).
Ability of AGT mutant proteins to affect survival and mutagenesis in cells
treated with MNNG
GWR109 cells containing AGT plasmids were grown in 10 ml of M9 media
supplemented with 50 µg/ml ampicillin, 50 µg/ml kanamycin and 0.3 mM
IPTG. Cultures were grown in a 37°C water bath shaken at 220 r.p.m. to an
A600 of 0.7. The cultures were pelleted and resuspended in 5 ml M9 media
and treated with 0–4 µg/ml MNNG (dissolved in 100 mM sodium acetate
buffer, pH 5.0) at 25°C for 1 h. The cells were centrifuged at 3000 r.p.m.,
suspended in 1 ml of M9 salts. Dilutions ranging from 1:100 to 1:10 000
were made and plated on M9 plates supplemented with 0.2% glucose, histidine
(40 µg/ml), ampicillin (50 µg/ml) and kanamycin (50 µg/ml) and incubated
at 37°C for 1–2 days to estimate the number of surviving colonies. In order
to assess his1 revertants, cells were plated on M9 media plates lacking
histidine. The mutation frequency was calculated as the number of his1
revertants/108 survivors. The incidence of Lac1 revertants in response to
MNNG in the strains FC218, FC326 and CJM2 were measured under the
same conditions using growth on galactose as described (37,46).
Results
When the inactive human AGT mutant C145A was expressed
in the his– ada– ogt– E.coli strain GWR109 (36), there was a
Expression of the inactive C145A mutant
Fig. 3. Effect of C145A AGT protein on effects of MNNG on G:C→A:T transition mutations in strains competent and deficient in NER. (a) and (b) Results
for the induction of G:C→A:T transition mutations in response to MNNG. (c) and (d) Effect of MNNG on cell killing. Results for the strain FC218 (open
symbols) which is competent in NER are shown in (a) and (c) and results for strain CJM2 which lacks NER (closed symbols) are shown in (b) and (d).
Results are shown for cells expressing wild-type AGT (inverted triangles), no AGT protein (triangles), C145A mutant AGT (circles) and SAMDC (squares).
The values that were statistically different (P , 0.01) from the cells expressing SAMDC used as a control are marked with asterisks. Results are means 6
SD for at least four estimations.
Fig. 4. Effect of C145A AGT and wild-type AGT protein on effects of
MNNG on A:T→G:C transition mutations. Mutations were measured in
strain FC326 using cells expressing wild-type AGT (inverted triangles),
C145A mutant AGT (circles) and SAMDC (squares). The values that were
statistically different (P , 0.01) from the cells expressing SAMDC used as
a control are marked with asterisks. Results are means 6 SD for at least
four estimations.
clear decrease in survival and an increase in mutations leading
to a reversion to the ability to grow without histidine after
treatment with MNNG. In order to confirm that this difference
was due to the C145A protein, the cells expressing mutant
C145A were compared with cells expressing a similar amount
of an irrelevant protein SAMDC, an inactive E11Q mutant of
the enzyme, S-adenosylmethionine decarboxylase (40), or to
cells expressing another inactive AGT mutant Y114E
(Figure 1). As shown in Figure 1a, the cells expressing either
SAMDC or Y114E gave a similar number of MNNG-induced
mutations to the control cells that contained the pIN vector
plasmid without an insert but C145A gave an ~2-fold increase
in mutations. As expected, the expression of the wild-type
AGT almost completely protected the cells from these
mutations (Figure 1a) or from killing (Figure 1b). The presence
of the C145A mutant increased the killing of GWR109 cells
by MNNG when compared with either Y114E or SAMDC
(Figure 1b).
The C145A mutant AGT is known to bind to DNA containing
O6-methylguanine (32) whereas the Y114E mutant is deficient
in DNA binding (42). The results can therefore be explained
if the C145A binds to the methylated DNA produced by
MNNG and prevents repair by other pathways. In order to test
this hypothesis, a double mutant Y114E/C145A to disrupt the
DNA binding region of the C145A mutant was made. When
this protein was expressed in GWR109 cells, the level of AGT
protein formed was the same as wild-type and the C145A
(Figure 2). However, the cells expressing the Y114E/C145A
mutant AGT protein did not show the increase in mutations
reverting to the his1 phenotype or the increase in killing
brought about by the C145A single mutation (Figure 1). These
results suggest that the binding of the C145A mutant to
methylated DNA is responsible for the increase in MNNGmediated toxicity.
Since the majority of the mutations produced by MNNG
are due to O6-methylguanine, it would be expected that the
C145A mutant AGT increases G:C→A:T transitions. This was
confirmed using the strain FC218, which has a point mutation
at the Glu-461 codon that renders it Lac– but can be reverted
to the Lac1 phenotype by a G:C→A:T transition (37,46). As
105
S.Edara, S.Kanugula and A.E.Pegg
shown in Figure 3a, the C145A mutant AGT increased the
frequency of MNNG-induced Lac1 revertants in this strain
supporting the hypothesis that these mutations are due to an
increased persistence of O6-methylguanine in the DNA. The
expression of wild-type AGT greatly reduced the production
of Lac1 revertants by MNNG in this strain (Figure 3a).
It is known that O6-methylguanine is a substrate for nucleotide excision repair (NER) in E.coli (47,48). As previously
reported (38), strain CJM2, which is similar to FC218 but also
has a deficiency in NER, was more sensitive to both killing
(Figure 3c and d) and reversion to the Lac1 phenotype (Figure
3a and b) when treated with MNNG. The expression of the
wild-type AGT decreased the incidence of mutations and the
killing of CJM2 cells by MNNG. However, the expression
of C145A mutant AGT did not increase the toxicity and
mutagenicity of MNNG in this strain (Figure 3b and d). These
results suggest that the binding of the C145A mutant to DNA
containing O6-methylguanine prevent its repair by NER.
Another DNA methylation product that has been reported
to be a substrate for AGT is O4-methylthymine (15,19,49–51).
O4-Methylthymine is a very minor component of the total
methylation damage (52) but is highly mutagenic causing
A:T→G:C transition mutations (5,53).These mutations can be
assayed by reversion to the Lac1 phenotype using strain FC326
(37). However, as shown in Figure 4, we were able to confirm
the report by Samson et al. (38) that the presence of wildtype AGT does not reduce the frequency of the A:T→G:C
transition mutations produced by MNNG but, instead, actually
increases this frequency. The C145A mutant also increased
the mutation frequency. This increase was slightly less than
that produced by wild-type AGT but the difference between
wild-type and C145A mutant AGT was not statistically significant.
Discussion
The most plausible explanation for the increase in MNNGmediated mutations and cell killing produced by the presence
of the C145A mutant AGT is that this protein binds to O6methylguanine sufficiently tightly to prevent its repair by other
DNA pathways. This interpretation is supported by several
different results: (i) the increase in mutations was clearly due
to a greater persistence of O6-methylguanine in DNA since
mutations were seen in the FC218 strain which requires a
G:C→A:T transition to revert to Lac1; (ii) the effect was not
seen with the Y114E mutant of AGT, which is also inactive
but fails to bind to DNA (42,54), and incorporation of the
Y114E mutation into the C145A mutant AGT abolished its
ability to increase MNNG toxicity; and (iii) the effect of
the C145A mutant AGT was not seen in the CJM2 strain
lacking NER.
These results provide further support for experiments showing that NER is able to recognize O6-methylguanine despite
the minimal distortion of the DNA structure produced by this
small lesion (47,48,55). Although alkyltransferase-catalyzed
repair is the predominant route for removal of this lesion,
NER provides a back-up pathway for cells with limited
amounts of alkyltransferase. However, in order for NER to be
efficient, any interference by inactive AGT must be prevented.
After repair of O6-methylguanine by AGT, the protein is
converted to an inactive form with S-methylcysteine at the
active site. Our results suggest that a mechanism to ensure
that this protein does not continue to bind to DNA containing
106
O6-methylguanine must exist in order to allow repair by other
pathways.
The fate of the methylated form of the AGT protein that
results from repair of DNA is not fully understood but several
studies have shown that the protein undergoes a conformational
change after alkylation of the active site cysteine (56–58) and
is degraded rapidly (59,60). There is also evidence that the
alkylated form of the protein becomes ubiquitinated (60,61).
All of these changes could help to prevent the binding of the
inactive form of the AGT to remaining lesions in the DNA
and thus avoid the toxic effects that we observe with the
C145A mutant AGT.
The repair of O4-methylthymine in DNA by mammalian
AGTs has been the subject of some controversy. Although it
has been well established for some time that certain microbial
alkyltransferases can repair O4-methylthymine (15,19,49),
repair of this lesion by extracts from mammalian cells could
not be demonstrated in several laboratories (62). However,
work carried out with recombinant AGTs, which in some cases
were present in great excess over the amount of substrate,
showed that even human AGT could bring about this repair
(50). Detailed studies have indicated that the efficiency with
which O4-methylthymine is repaired by AGTs varies considerably according to the source of the repair protein and repair
by the human AGT is very slow, in particular when compared
with certain microbial AGTs (18,49,51). Although this work
is of considerable interest with respect to the information
provided concerning the active site of alkyltransferases, it does
not answer the question of whether human AGT is effective
in repairing this lesion in vivo. Our results using strain FC326
to measure A:T→G:C transition mutations arising from O4methylthymine confirm the report of Samson et al. that the
presence of mammalian AGTs in E.coli actually increases the
incidence of such mutations in response to MNNG (38).
Therefore, it appears unlikely that AGT plays a significant role
in removing the minor methylation product O4-methylthymine
from DNA in human cells. It is noteworthy that recent studies
have shown that even in E.coli, mutations arising from plasmids
containing O4-methylthymine were not reduced when measured
in alkyltransferase deficient strains (5).
In the case of O4-methylthymine-mediated mutations, both
the wild-type and the C145A mutant human AGT increase the
number of mutations (Figure 4). This is consistent with the
proposal that wild-type AGT repairs O4-methylthymine so
slowly that it interferes with repair of the lesion by NER (38).
Our results are in agreement with this hypothesis and show
that, when a totally inactive AGT protein is used, the O6methylguanine-induced G:C→A:T transition mutations and
cell killing by AGT are also increased. The small difference
in the A:T→G:C transition mutation frequency between the
cells expressing wild-type AGT and the cells expressing
C145A (Figure 4) was not statistically significant but could
indicate that the C145A mutant does not bind quite so tightly
to O4-methylthymine lesions as the wild-type AGT.
Acknowledgements
We are most grateful to Dr L.Samson for the gifts of the bacterial strains used
in this work. This work was supported by the National Cancer Institute with
grants CA-18137, CA-71976 and CA-57725.
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Received July 22, 1998; revised September 2, 1998;
accepted September 3, 1998