[CANCER RESEARCH 53, 4750-4753, October 15, 1993]
Advances in Brief
A Single Amino Acid Change in Human 06-Alkylguanine-DNA
Alkyltransferase
Decreasing Sensitivity to Inactivation by O6-Benzylguanine1
Tina M. Crone and Anthony E. Pegg
Departments of Cellular und Molecular
Hershey, Pennsylvania ! 7033
Physiology
and of Pharmacology,
Milton
S. Hershey
Center, Pennsylvania
State
University
College
of Medicine,
zylcysteine in the AGT and the formation of stoichiometric amounts
of guanine following incubation with O6-benzylguanine.3 There is
Abstract
Mammalian
O'-alkylguanine-DNA
alkyltransferases
readily inactivated by incubation with the pseudosubstrate,
(AGTs) are
O6-benzylgua-
nine, but the equivalent protein from the Escherichia coli ogt gene is much
less sensitive and the Saccharomyces cerevisiae and E. coli oda gene prod
uct AGTs are completely resistant to this compound. We have expressed
the normal human ACT and various point mutations (C145A, W100A,
and P140A) in an u</u ogt~ strain of E. coli and tested these proteins
against DNA substrates containing 06-methylguanine, for inactivation by
06-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue
forms the methyl acceptor site. Mutants VS1(10A and P140A were fully
active against methylated DNA substrates but the P140A mutant was
much less sensitive to inactivation by 06-benzylguanine and failed to form
significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]guanine. The proline at position 140 in mammalian AGTs is replaced by
alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. These
results suggest that this proline residue affects the configuration of the
active site allowing the 06-benzylguanine
to enter and react with the
mammalian AGT. The production of resistance to 06-benzylguanine by a
single base change raises the possibility that such resistance may arise
quite readily in cells of tumors treated therapeutically with the combina
tion of 06-benzylguanine and an alkylating agent.
considerable similarity between the human AGT and the AGT proteins
isolated from microorganisms and the sequence surrounding the cys
teine acceptor site is identical (3, 4, 8). However, regardless of this
similarity, the AGT proteins derived from carboxyl terminal fragment
of the Escherichia coli ado. gene (9) and the Saccharomyces cerevisiae
AGT3 were found to be completely resistant to inactivation by Ohbenzylguanine and the AGT derived from the Escherichia coli ogt
gene was much less sensitive.3 These results suggest that there is some
difference in the active site of these AGT proteins and show that the
microbial proteins are not good models for the design of inactivators
of the mammalian AGT. Furthermore, they raise the possibility that
resistance to O6-benzylguanine might arise in mammalian cells treated
with this AGT inactivator and a toxic and mutagenic alkylating agent.
We have examined this question by expressing the human AGT and
defined mutants of it in E. coli. We now report a single amino acid
change that bestows a significant level of resistance to O6-benzylguanine in the human AGT.
Materials and Methods
Introduction
AGT2 is a DNA repair protein that plays an important role in
protecting cells from the toxic effects of monofunctional alkylating
agents and chloroethylating drugs (1-4). AGT has a unique mecha
nism of action in that it brings about the transfer of alkyl groups
present on the O''-position of guanine in DNA to a cysteine residue
Materials, Bacterial Cells, and Plasmids. GWR109 cells (10) were ob
tained from Dr. Leona Samson, Department of Molecular and Cellular Toxi
cology, Harvard School of Public Health, Boston, MA. DH5a MCR cells were
purchased from Bethesda Research Laboratories (Gaithersburg, MD). BamHI
was purchased from GIBCO BRL (Gaithersburg, MD). EcoRI and T4 DNA
ligase were purchased from New England Biolabs (Beverly, MA). Ampicillin,
kanamycin, MNNG, and IPTG were purchased from Sigma Chemical Com
pany (St. Louis, MO). CJ236 cells, plasmid pGem-3Zf( + ), and helper phage
M13K07 were purchased from Promega Corporation (Madison WI). Plasmid
pINAGT, which expresses the human AGT in E. coli, was produced by insert
ing the human cDNA sequence (8) into the E. coli expression vector pINIIIA3(lppp"5) (11) using the £coRIand BamH\ sites in the vector and polymerase
located within the AGT amino acid sequence (5, 6). The resulting
S-alkylcysteine in the protein is not converted back to cysteine. There
fore, the AGT can act only once and the number of O6-alkylguanine
residues that can be repaired is equal to the number of available AGT
molecules. Tumor cells expressing high levels of AGT are resistant to
killing by therapeutic methylating or chloroethylating drugs and such
inherent resistance may limit the clinical effectiveness of these agents
(1-4, 6). There is, therefore, considerable interest in the synthesis of
compounds that would block the AGT activity and thus enhance their
action. Recent work has indicated that O6-benzylguanine may be
suitable for this purpose. O6-Benzylguanine was found to be a very
potent time and concentration dependent inactivator of the human
alkyltransferase (7). Inactivation was irreversible suggesting that O6benzylguanine acts as an alternative substrate for the protein. This
mechanism has now been confirmed by the identification of S-benReceived 7/15/93; accepted 9/15/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants CA-18137 and CA-57725 from the National
chain reaction to generate the appropriate sites in the cDNA. The means of
construction changes the amino terminal sequence by the addition of 5 amino
acids giving a sequence MKGGIL- in place of M-.
Site-directed Mutagenesis. pINAGT was digested with £coRIand BamHI
and the resulting piece which contains the human AGT amino
sequence was inserted into pGem-3Zf(+) which was used for
mutagenesis using the following oligodeoxynucleotides
and the
tide-directed Mutagenesis System 2.1 kit (Amersham, Arlington
according to manufacturer's instructions. Oligodeoxynucleotides
acid coding
site directed
OligonucleoHeights, IL),
were synthe
sized in the Macromolecular Core Facility, Hershey Medical Center, by using
a Milligen 7500 DNA synthesizer. The following sense strands were synthe
sized, mismatches underlined, to produce the amino acid changes of tryptophan
100 to alanine, proline 140 to alanine, and cysteine 145 to alanine, respec
tively: 5'-CCAGACAGGTGTTAGCAAAGCTGCTGAAG-3';
5'-GGCAATCCTGTCGCCATCCTCATCCCG-3';
and 5'-CCATCCTCATCCCGGCCCACAGAGTGGTC-3'. After screening to identify the desired mutants, the inserts
were transferred back into the pIN expression vector. All mutants were verified
by sequencing the entire AGT amino acid coding region.
Cancer Institute.
2 The abbreviations used are: AGT, O^-alkylguanine-DNA alkyltransferase (EC 2.1.
63); MNNG, A/-methyl-A/'-nitro-7v'-nitrosoguanidine; IPTG, isopropyl-ß-o-thiogalactopyranoside; cDNA, complementary
Medical
3 A. E. Pegg, M. Boosalis, L. Samson, R. C. Moschel, T. L. Byers, K. Swenn, and M.
E. Dolan, submitted for publication.
DNA.
4750
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RESISTANCE
TO O'-BENZYLGUANINE
MNNG Survival Assay. GWR109 cells containing the pINAGT plasmid or
mutants were grown overnight in 5 ml of LB broth containing 50 fig/ml
ampicillin, 5Ü/¿g/mlkanamycin, and 0.3 ITIM1PTG. Cultures of 5 ml of the
same medium were inoculated with 50 /xl from the overnight cultures and
grown in 50-ml conical tubes agitated at 150 rpm in a 37°Cwater bath. MNNG
at concentrations ranging from 0 to 20 fig/ml was added when the Em> reached
0.7 using a stock MNNG stock solution made immediately before use at a
concentration of 10 mg/ml in 20% dimethyl sulfoxide. Cultures were agitated
at 250 rpm in a 25°Cwater bath for 30 min. Afterwards, dilutions were plated
on LB agar plates containing 0.3 mm IPTG in addition to ampicillin and
kanamycin as described above. Plates were incubated at 37°Cand surviving
colonies were counted 16-20 h later.
Measurement of ACT Activity and Protein. Cell containing the pINAGT
or mutant plasmids were grown as described above through log phase until
f HH)reached 1.0. Extracts were made and assayed for AGT activity by assaying
the loss of O6-[3H]methylguanine from a 3H-methylated DNA substrate as
described previously (7). AGT protein amount and size was determined by
sodium dodecyl sulfate-polyacrylamide
gel electrophoresis separation fol
lowed by immunoblotting using antibodies to peptide sequences near the
amino terminus of the AGT and scanning of the bands with a laser densitometer (12). Inactivation of the AGTs by O"-benzylguanine or O''-alIylguanine
was determined (7, 13) by incubating the AGT with the inhibitor for 30 min at
37°Cprior to adding the 3H-methylated DNA substrate.
For purification of the control, W100A, and P140A alkyltransferases, the
cells containing pINAGT were grown to an /4HK)of 1.7-1.8 in l^t liters of LB
broth with 50 /ig/ml ampicillin and 0.15 ITIMIPTG. The cells were pelleted at
4000 X g for 10 min at 4°C,resuspended in LB broth, and pelleted again. All
subsequent operations were carried out at 4°C.The pellets were resuspended
in 20 iriMTris-HCl, pH 8.5-1 HIMEDTA-3 HIMdithiothreitol-0.4
M NaCl and
the cells were broken with a French Press. Insoluble debris was pelleted at
90,000 X g for 30 min and the supernatant was saved. Polymin P (in 5%
solution, pH 8.0) was added to give 0.36 /¿gPolymin P/juig DNA and the
mixture was centrifuged at 15,000 X g for 15 min. The supernatant was taken
and made 40% saturated in ammonium sulfate. After 30 min, the precipitated
protein was removed by centrifugation at 15,000 x g for 15 min, more
ammonium sulfate was added to obtain 55% saturation. After 30 min, the
precipitated protein was collected at 15,000 X g for 15 min and dissolved in
Buffer A [50 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid pH 8.0,
containing 1 ITIMEDTA and 3 ITIMdithiothreitolj. After desalting by passage
through a PD-10 desalting column (Pharmacia) equilibrated with Buffer A, the
solution was fractionated by cation exchange chromatography using a Mono S
HR 10/10 fast protein liquid chromatography column (Pharmacia) at room
temperature. After loading at a flow rate of 2 ml/min, the column was washed
with Buffer A at 4 ml/min until the A^,, returned to base line. Then, the column
was eluted with a gradient of 0 to 1 M NaCl in Buffer A such that 0.3 M NaCl
was reached within 40 min. The alkyltransferase activity eluted at about 0.15
M NaCl. The fractions corresponding to this peak were pooled and concen
trated. The final preparations gave a single major band of the expected mo
lecular weight and had a specific activity of about 30,000-32,000 pmol/mg
which is about 75% of the theoretical value and is comparable to results in
other reports (14).
Formation of Guanine from O6-Benzylguanine. O''-Benzyl[8-3H]guanine was produced by Amersham Corporation (Arlington Heights, IL) by
catalytic tritium exchange of O6-benzylguanine with tritiated water and was
purified by high performance
liquid chromatography
on a Beckman Ultra-
C145A, W100A, and P140A. The first two of these mutants are
formed at residues that are conserved in all known AGT sequences.
The cysteine residue at position 145 is that forming the alkyl acceptor
site. The tryptophan at position 100 was mutated because of the
possibility that it may be involved in DNA binding and the proline at
position 140 was changed because this residue is present in all mam
malian AGTs but is not present in the microbial AGTs that are resistant
to O6-benzylguanine. The control and mutant AGT proteins were
expressed in GWR109 cells that lack endogenous AGT activity (10).
All three of these mutant AGT proteins were expressed at similar
levels to the wild type protein as indicated by Western blots developed
with an antibody to a peptide sequence corresponding to a region close
to the amino terminus of the human AGT (Table 1). The only band
recognized by this antibody had the expected molecular weight of
about 22,000 and this band was completely absent from the host E.
coli cells (results not shown). The small variation in levels (Table 1),
which is within a factor of 2, is probably accounted for by small
differences in growth conditions and by the semiquantitative method
of the assay using immunoblotting. Two tests were used to check the
AGT activities of these mutants. Extracts were made and assayed for
the ability to bring about a loss of Oil-[-1H]methylguanine from a
3H-methylated DNA substrate. This showed that mutation C145A
completely inactivated the AGT whereas mutations W100A and
P140A had little or no effect on the ability to repair methylated DNA
(Table 1). This result was confirmed by studying the effect of these
plasmids on the survival of GWR109 cells treated with MNNG (Fig.
1). The GWR109 cells were very sensitive to killing by this methylating agent and the expression of control human AGT rendered the
cells resistant. The expression of C145A AGT did not increase sur
vival but both plasmids containing AGT with the W100A and P140A
mutations showed the same protective effect as the control AGT.
The effect of O''-benzylguanine on the activity of these mutant
AGTs was first examined using crude extracts of the cells expressing
the activity. These extracts were incubated for 30 min with varying
concentrations of O''-benzylguanine and the remaining AGT activity
was measured after addition of the 3H-methylatcd DNA substrate. As
shown in Fig. 2A, there was no difference in the sensitivity of the
W100A mutant compared to the wild type but the P140A mutant was
much more resistant to 06-benzylguanine. The level of O''-benzylguanine needed to produce a 50% inactivation was increased from 0.2
JU.M tO 8 /XM.
In order to confirm this result, the P140A mutant and control AGT
proteins were examined for the ability to form guanine from O6benzylguanine (Fig. 3). The control AGT showed the expected result
with guanine formation occurring in a time and concentration depen
dent fashion until all of the Oh-benzyl[8-JH]guanine was converted
but the P140A had only a very weak activity in producing guanine.
These results establish unequivocally that the P140A mutation
greatly reduces the ability of the human AGT to interact with
Ofi-benzylguanine.
sphere octadecylsilone column (25 cm X 4.6 mm) using isocratic elution at a
temperature of 35°Cand a buffer of equal parts methanol and 0.05 M ammo
nium formate, pH 4.5. Measurements of guanine formation from O6-benzylguanine were carried out using various amounts of the O''-benzyl[8-3H]gua-
Table 1 AGT activity in bacterial extracts from GWR109 cells expressing control,
C145A, W10UA,and P140A mutations
activity (pmol (}''protein (units"/
methylguanine removed/
mg soluble
protein)<0.1<0.1
mg soluble
protein)<189
PlasmidNoneC145A
nine and alkyltransferase protein in an assay buffer consisting of 50 ITIM
Tris-HCl (pH 7.5), 0.1 ITIMEDTA, and 5 IHMdithiothreitol. The formation of
labeled product was stopped by the addition of 0.6-0.8 ml of the same buffer
containing 0.2 ITIMguanine and 0.2 ITIMO''-benzylguanine. Aliquots were then
separated by HPLC as described above.
120±31fc83
8457100
±7117
±32AGT
AGTAGT
" The content of AGT protein was determined by dcnsitometric scanning of Western
blots and the amount present in the cells carrying the pINAGT control was set at 1(H).
h Mean ±SD.
P140AW1ÃœOAControl
Results and Discussion
The human AGT cDNA sequence was inserted into an E. coli
expression vector forming pINAGT. Various mutants were produced
and inserted into this vector as described above. These included
4751
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RESISTANCE
100
TO 0"-BENZYLGUAN1NE
-
O
5
10
15
MNNG (ng/ml)
Fig. 1. Effect of plasmids expressing mutant AGTs on survival of GWR109 cells
treated with MNNG. The cells were grown under conditions leading to expression of AGT
and treated with MNNG at the concentrations shown, and the surviving cells were
determined as described in "Materials and Methods." Results are shown for GWR109
cells alone and for GWR109 cells containing plasmids expressing control AGT, mutant
W100A AGT. mutant C145A AGT. and mutant PI40A AGT.
Since the P140A AGT is highly active in repairing methylated DNA
in vitro and in protecting cells from MNNG, it is likely that this
mutation has little effect on the ability of the AGT to react with
methylated DNA but it is possible that the rate of repair is slowed.
Since this rate is normally very rapid, it is hard to measure accurately.
Therefore, in order to test whether the P140A mutation has a selec
tively greater effect on the reaction with 06-benzylguanine, a com
petition assay was carried out in which O6-benzylguanine was added
directly without preincubation to the assay medium containing 3Hmethylated DNA (Fig. 2B). Although the difference was slightly less
than in the inactivation assay shown in Fig. 2A, the P140A mutant was
still much more resistant to competitive inactivation by 06-benzylguanine with the concentration needed to produce 50% inactivation
increasing from 1.1 /IM to 13 /AM(Fig. 2B). The W100A mutant AGT
showed similar sensitivity to wild type in this assay.
Human AGT (13) and the E. coli Ogt ACT3 are both sensitive to
inactivation by O6-allylguanine. As shown in Fig. 2C, the P140A
mutant AGT was slightly resistant to this inactivator when compared
to the control AGT but the difference was only 2.8-fold with the
concentration needed for 50% inactivation increasing from 20 /¿M
to
56 JIM, much less than with C^-benzylguanine. Since the allyl sub
stituent is smaller than benzyl, this is consistent with the hypothesis
that the major factor underlying the species specificity of AGT inac
tivation by these pseudosubstrates is the size of the active site limiting
the access of the compounds.
The fact that the mutation conferring resistance to 06-benzylguanine is that of a proline residue is also in agreement with this hypoth
esis. Proline residues are known to constrain the conformation of
adjacent residues and can lead to kinks in a-helices (15, 16). Such
bends can contribute to conformational shifts accommodating sub
strate binding. The proline at position 140 is located at the amino
terminus of the peptide sequence—ILIPCHRV—which forms the ac
ceptor site of the AGTs and is very highly conserved (3, 4). It is,
therefore, quite probable that this proline residue results in a bend or
conformational change that increases the size of the space surrounding
the active site. In the yeast AGT and the Ada AGT that are insensitive
to 06-benzylguanine, this proline is replaced by an alanine as in the
mammalian mutant we describe. In the Ogt AGT, which shows some
sensitivity to 06-benzylguanine but is much less susceptible than the
human AGT, this proline is replaced by a serine but there is another
proline located two residues earlier in this sequence.
Studies of mutant proteins produced by site directed mutagenesis
may be compromised by the mutation producing a major distortion in
the protein structure. Such changes can lead to partial denaturation
and a dramatic reduction in the activity and stability of the protein. We
have observed that a number of mutations in the human AGT includ-
0
Fig. 2. Effect of O6-benzylguanine
50
100 150
UM O'-Allylguanine
200
and 06-allylguanine
on AGT activity. In the ex
periment shown in A, control AGT. mutant WIOOA AGT, or mutant P140A AGT were
incubated at 37°Cwith O6-benzylguanine for 30 min in 50 mm Tris-HCl (pH 7.5), 1 rrun
dithiothreitol, and 0.1 mu EDTA in a total volume of 0.5 ml. The 'H-methylaled DNA
substrate was then added and the remaining AGT activity was measured by determining
the extent of loss of O6-['H]methylguanine from this substrate in a further incubation for
30 min. The results are expressed as the percentage of the activity found when no
O6-benzylguanine was added. The total activity declined by less than 5% during incuba
tion for 30 min at 37°Cin the absence of the inhibitor. In the experiment shown in B,
control AGT. mutant WIOOA AGT, or mutant P140A AGT were added to an assay mix
containing a 'H-melhylated DNA substrate, the O6-benzylguanine concentration shown,
and 50 mM Tris-HCl (pH 7.5), 1 mM dilhiolhreitol, and 0.1 ITIMEDTA in a total volume
of 1 ml. The mixture was incubated at 37°Cfor 30 min and the inhibition of AGT activity
was measured by determining the extent of removal of O6-['H]methylguanine
from the
DNA. The results are expressed as the percentage of the activity found when no O6benzylguanine was added. In the experiment shown in C, control AGT or mutant PI40A
AGT were incubated at 37°Cwith O6-allylguanine for 30 min prior to the addition of the
JH-melhylaled DNA substrate. Other details were the same as for A.
ing changes of H146, R147, and truncations of the protein at either the
amino or carboxyl termini do lead to a marked instability of the AGT
protein which is indicative of such distortion or incorrect folding.4
Ling-Ling et al. (17) have also noted that mutations in the area
surrounding the active site leads to an apparent instability of the
protein. However, the mutant AGTs used in the experiments described
in this paper do not appear to present problems in this respect. All
were expressed to levels comparable to the wild type AGT (Table 1),
the P140A and WIOOA mutant AGTs protected GWR109 cells from
killing by MNNG to the same extent as the control AGT (Fig. 2), and
the half-life of these proteins in E. coli was >24 h (results not shown).
Furthermore, the P140A mutant that is of particular interest was
readily purified by the same procedure as used for the control AGT
and was not separable from the control by any of the Chromatographie
steps used. Finally, the experiment shown in Fig. 2B shows clearly that
this P140A mutant AGT has a reduced capacity to interact with
* T. M. Crone, L. Wiest, and A. E. Pegg, unpublished observations.
4752
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RESISTANCE
TO O*-BENZYLGUANINE
vation are clearly needed along with the determination of the three
dimensional structure of the protein. Diffraction patterns have been
obtained from crystals of the Ada AGT (20). Major consideration also
needs to be given to the design of inactivators of the AGT using
O6-substituents of smaller size that could not be excluded by steric
factors.
Acknowledgments
We thank Dr. R. C. Moschel for the synthesis of O6-benzylguanine and
O^-allylguanine, Dr. L. Samson for bacterial strains and advice, M. E. Dolan
for advice, and L. Wiest and K. Swenn for technical assistance.
S 30000
References
I 20000
3
m 10000
I
°
0
20
40
60
|ig Alkyltransferase (AGT)
Fig. 3. Formation of guanine from O^-benzylguanine by control and P140A mutant
AGTs. In the experiment shown in A, a total volume of 0.2 ml of 50 HIMTris-HCl, pH
7.5-0.1 HIMEDTA5 mu dithiothreitol containing 540 pmol of O6-benzyl[8-3H]guanine
(40,000 cpm) and 10 jig of AGT was incubated at 37°Cfor the time shown. The amount
of PHJguanine was then determined as shown for the control AGT or P140A mutant AGT.
In the experiment shown in B, a total volume of 0.2 ml of 50 mMTris-HCl pH 7.5-0.1 mM
EDTA 5 mM dithiothreitol containing 540 pmol of O6-benzyl[8--'H]guanine (40,000 cpm),
and varying amounts of the AGT was incubated at 37°Cfor 30 min. The amount of
pHJguanine was then determined as shown for the control AGT or P140A mutant AGT.
O6-benzylguanine
even in the presence of a methylated DNA sub
strate; therefore the resistance to inactivation cannot be attributed
solely to a slower rate of reaction with all substrates.
Pretreatment of nude mice with O6-benzylguanine enhances the
response of human tumor xenografts expressing AGT to therapeutic
methylating and chloroethylating agents (4, 18, 19). Clinical trials of
such combination therapy are being planned. At present, the stability
and activity of the P140A mutant AGT in mammalian cells are not
known but experiments are under way to express this protein in tumor
cells and determine the extent to which these cells are refractory to
this combination chemotherapy. However, our results suggest very
strongly that the development of resistance via the appearance of cells
containing mutant AGTs that are no longer sensitive to O6-benzylguanine may be a major problem if such combination therapy is
prolonged. The change of P140A involves only a single base substi
tution and the alkylating agents are known mutagens. Other mutations
may also impart resistance to O6-benzylguanine. For example, the E.
coli Ada AGT and the yeast AGT are even more resistant than the E.
coli Ogt protein or the human P140A AGT. There is a proline at
position 138 in the human AGT sequence which is present in the Ogt
but not in the yeast or Ada proteins. Also, the microbial AGTs are
truncated at the carboxyl end compared to the mammalian equiva
lents. Either of these sites could also be involved in resistance to
O6-benzylguanine. It should also be noted that the AGT protein prob
ably undergoes a conformational change on binding to DNA. This
change may modify the active site and we have found that the rate of
conversion of O6-benzylguanine into guanine by the mammalian AGT
is increased about 3.5-fold by the presence of calf thymus DNA.4
Further studies on the relationship between structure and this inacti
1. Brent, T. P. Isolation and purification of CX'-alkylguanine-DNA alkyltransferase from
human leukemic cells. Prevention of chloroethylnitrosourea-induced
cross-links by
purified enzyme. Pharmacol. Ther., 31: 121-140, 1985.
2. Ludlum, D. B. DNA alkylation by the haloethylnitrosoureas: nature of modifications
produced and their enzymatic repair or removal. Mutât.Res., 233: 117-126, 1990.
3. Pegg, A. E., and Byers, T. L. Repair of DNA containing O6-alkylguanine. FASEB J.,
6: 2302-2310, 1992.
4. Mitra, S., and Kaina, B. Regulation of repair of alkylation damage in mammalian
genomes. Prog. Nucleic Acid Res. Molec. Biol., 44: 109-142, 1993.
5. Lindahl, T, Sedgwick, B., Sekiguchi, M., and Nakabeppu, Y. Regulation and expres
sion of the adaptive response to alkylating agents. Annu. Rev. Biochem., 57:133-157,
1988.
6. Pegg, A. E. Mammalian 06-alkylguanine-DNA alkyltransferase: regulation and im
portance in response to alkylating carcinogenesis and therapeutic agents. Cancer Res.,
50: 6119-6129, 1990.
7. Dolan, M. E., Moschel, R. C., and Pegg, A. E. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by Oft-benzylguanine provides a means to
evaluate the role of this protein in protection against carcinogenic and therapeutic
alkylating agents. Proc. Nati. Acad. Sci. USA, 87: 5368-5372, 1990.
8. Taño,K., Shiota, S., Collier, J., Foote, R. S., and Mitra, S. Isolation and structural
characterization of a cDNA clone encoding the human DNA repair protein for Ohalkylguanine. Proc. Nati. Acad. Sci. USA, 87: 686-690, 1990.
9. Dolan, M. E., Pegg, A. E., Dumenco, L. L., Moschel, R. C., and Gerson, S. L.
Comparison of the inactivation of mammalian and bacterial C^-alkylguanine-DNA
alkyltransferases by Ofi-benzylguanine. Carcinogenesis (Lond.), 12: 2305-2310,
1991.
10. Rebeck, G. W., and Samson, L. Increased spontaneous mutation and alkylation
sensitivity of Escherichia coli strains lacking the ogi O"-methylguanine DNA repair
methyltransferase. J. Bacteriol., 173: 2068-2076, 1991.
11. Duffaud, G. D., March, P. E., and Inouye, M. Expression and secretion of foreign
proteins in Escherichia coli. Methods Enzymol., 153: 492-507, 1987.
12. Pegg, A. E., Wiest, L., Mummert, C., and Dolan, M. E. Production of antibodies to
peptide sequences present in human 06-alkylguanine-DNA alkyltransferase and their
use to detect this protein in cell extracts. Carcinogenesis (Lond.), 12: 1671-1677,
1991.
13. Moschel, R. C., McDougall, M. G., Dolan, M. E., Siine, L., and Pegg, A. E. Structural
features of substituted purine derivatives compatible with depletion of human O1'alkylguanine-DNA alkyltransferase. J. Med. Chem., 35: 4486-4491, 1992.
14. Gonzaga, P. E., Potter, P. M., Niu, T, Yu, D., Ludlum, D. B.. Rafferty, J. A., Margison,
G. P., and Brent, T. P. Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated
DNA. Cancer Res.,
52: 6052-6058, 1992.
15. Barlow, D. J., and Thornton, J. M. Helix geometry in proteins. J. Mol. Biol., 201:
601-619, 1988.
16. Williams, K. A., and Debet, C. M. Proline residues in transmembrane helices: struc
tural or dynamic role? Biochemistry, 30: 8919-8923, 1991.
17. Ling-Ling, C., Nakamura, T., Nakatsu, Y., Sakumi, K., Hayakawa, H., and Sekiguchi,
M. Specific amino acid sequences required for O6-methylguanine-DNA methyltrans
ferase activity: analyses of three residues at or near the methyl acceptor site. Carci
nogenesis (Lond.), 13: 837-843, 1992.
18. Friedman, H. S., Dolan, M. E., Moschel, R. C., Pegg, A. E., Felker, C. M., Rich, J.,
Bigner, D. D., and Schold, S. C. Reversal of nitrosourea resistance in medulloblastoma and glioblastoma multiforme. J. Nati. Cancer Inst., 84: 1926-1931, 1992.
19. Mitchell, R. B., Moschel, R. C., and Dolan, M. E. Effect of O*-benzylguanine on the
sensitivity of human tumor xenografts to l,3-bis(2-chloroethyl)-l-nitrosourea
and on
DNA interstrand cross-link formation. Cancer Res., 52: 1171-1175, 1992.
20. Moody, P. C. E., Cutfield, S. M., Moore, M. H., and Dodson, G. G. Progress on the
structure determination of O"-methylguanine-DNA
methyltransferase from Esch
erichia coli. Acta Cryst., C-95, 1990.
4753
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A Single Amino Acid Change in Human O6-Alkylguanine-DNA
Alkyltransferase Decreasing Sensitivity to Inactivation by O6
-Benzylguanine
Tina M. Crone and Anthony E. Pegg
Cancer Res 1993;53:4750-4753.
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