From the Departments of Biochemistry and
¶ Chemistry, A. B. Hancock Jr. Memorial Laboratory for Cancer
Research, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer
Center, Vanderbilt University School of Medicine, Nashville, Tennessee
37232
Received for publication, September 29, 2000, and in revised form, December 4, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acrolein is produced extensively in the
environment by incomplete combustion of organic materials, and it
arises endogenously in humans as a metabolic by-product. Acrolein
reacts with DNA at guanine residues to form the exocyclic adduct,
8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a
correlation exists between HOPdG levels in Salmonella
typhimurium treated with acrolein and a resultant increase in
mutation frequency. Site-specifically modified oligonucleotides were
used to explore the mutagenic potential of HOPdG in Escherichia
coli strains that were either wild-type for repair or deficient
in nucleotide excision repair or base excision repair. Oligonucleotides
modified with HOPdG were inserted into double-stranded bacteriophage
vectors using the gapped-duplex method or into single-stranded
bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide
hybridization to establish the mutation frequency and the spectrum of
mutations produced by HOPdG. The correct base, dCMP, was incorporated
opposite HOPdG in all circumstances tested. In contrast, in
vitro lesion bypass studies showed that HOPdG causes
misincorporation opposite the modified base and is a block to
replication. The combination of these studies showed that HOPdG is not
miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.
The accumulation of DNA damage is believed to play a significant
role in genetic diseases, including cancer. An important form of DNA
damage results from the addition of electrophiles to form stable
adducts to the bases. In the case of bifunctional electrophiles,
cyclization can occur to form exocyclic adducts that obstruct
Watson-Crick base pairing (1). These adducts are anticipated to be
blocks to replication and, therefore, potential premutagenic lesions.
This has been demonstrated to be the case with the exocyclic adducts,
pyrimido[1,2- Acrolein, an
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
]purin-10(3H)-one
(M1G)1 and its
structural analogue,
1,N2-propano-2'-deoxyguanosine (PdG)
(2-4).
,
-unsaturated aldehyde, is an example of such a
bifunctional electrophile. Acrolein is produced exogenously as a
product of organic combustion and endogenously as a product of lipid
peroxidation (5-7). Acrolein also arises during cyclophosphamide metabolism (8). It is highly mutagenic to bacterial and mammalian cells
and exhibits tumor-initiating activity (9-11). The most likely mode of
action for acrolein-induced mutagenicity is its ability to form adducts
to DNA. The major adduct generated by the reaction of acrolein with
deoxyguanosine residues in DNA is 8-hydroxypropanodeoxyguanosine
(HOPdG), which is structurally related to M1G and PdG (Fig.
1) (12, 13). Acrolein has been shown to
be mutagenic in Salmonella typhimurium, and a correlation has been observed between HOPdG levels in S. typhimurium
treated with acrolein and the resultant increase in mutation frequency (14). The HOPdG adduct has been detected at relatively high levels in
DNA from healthy human tissues using 32P-postlabeling
combined with high performance liquid chromatography (15, 16).
View larger version (13K):
[in a new window]
Fig. 1.
Structures of exocyclic adducts.
A major obstacle in attempts to evaluate the mutagenic potential of
HOPdG has been the generation of oligonucleotides containing site-specifically incorporated HOPdG. The base lability of HOPdG prevents its direct incorporation into DNA by standard synthetic protocols. Recently, both Khullar et al. (17) and Nechev
et al. (18) described strategies for the stable,
site-specific incorporation of HOPdG into oligonucleotides using
oxidative generation of the aldehyde function after the base
deprotection step. This enables the construction of recombinant M13
genomes containing a single HOPdG adduct for use in mutagenesis assays
in Escherichia coli. In the present study, we prepared
double-stranded vectors containing HOPdG to evaluate its ability to
block replication, to induce mutations, and to be repaired. In
addition, single-stranded vectors were constructed to examine the
ability of HOPdG to induce mutations in the absence of repair or of
replication strand bias. The combination of these approaches revealed
that HOPdG does not induce mutations at a level of sensitivity well
below that needed to detect mutations caused by other structurally
related exocyclic adducts.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
T4 polynucleotide kinase, T4 DNA ligase,
deoxynucleotide 5'-triphosphates, BssHII, SacII,
EcoRI, and DNA polymerase I Klenow fragment
(exo) were purchased from New England BioLabs
(Beverly, MA). MOPS and calf thymus DNA were purchased from Sigma
Chemical Co. (St. Louis, MO). Tris-HCl (pH 8.0), EDTA, and sodium
dodecyl sulfate solution were obtained from Life Technologies, Inc.
(Grand Island, NY). Formamide was purchased from Aldrich Chemical Co.
Inc. (Milwaukee, WI). Ultrapure AquaPor LM agarose was from National
Diagnostics (Atlanta, GA). Nylon membranes (0.45 µm, 82 mm)
were from Schleicher & Schuell (Keene, NH). [
-32P]ATP
was purchased from PerkinElmer Life Sciences (Boston, MA). Bio-spin 6 columns were purchased from Bio-Rad (Hercules, CA). GELase enzyme was
from Epicentre Technologies (Madison, WI). Isopropyl
-D-thiogalactoside (IPTG) and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
were purchased from Gold Biotechnology Inc. (St. Louis, MO).
Oligonucleotides-- The unmodified 8-mer oligonucleotide for mutagenesis experiments, 5'-GGTGTCCG-3', was synthesized by Midland Certified Reagent Co. (Midland, TX). HOPdG-modified 8-mer oligonucleotides, 5'-GGT(HOPdG)TCCG-3', and 31-mer oligonucleotides, 5'-GACGAATTCGCGATC(HOPdG)TCGACTCGAGCTCAG-3', were synthesized using the method described by Nechev et al. (18). After purification, the purity of the HOPdG 8-mer and 31-mer oligonucleotides was checked by a 20% denaturing polyacrylamide gel. Both HOPdG-modified oligonucleotides were determined to be >99.5% pure by capillary gel electrophoresis.
The primers and all unmodified template oligonucleotides for in vitro bypass assays were prepared using an Applied Biosystems automated DNA synthesizer in the Vanderbilt University Molecular Toxicology Molecular Genetics Core. A 12-mer oligonucleotide used as a running start primer had the following sequence: 5'-CTGAGCTCGAGT-3'. The 13-mer standing start primer sequence was as follows: 5'-GAGCTCGAGTCGA-3'. 16-mer oligonucleotides were also synthesized with the following sequence: 5'-CTGAGCTCGAGTCGAN-3', where N refers to A, G, C, or T. Unmodified oligonucleotides were purified by electrophoresis on a denaturing 20% polyacrylamide gel. Oligonucleotide sizing markers (8-32 bases) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
Bacterial Strains and Vectors--
All bacteria strains used in
this study are derivatives of the E. coli strain AB1157
(thr-1, ara-14, leuB6,
(gpt-proA)62, lacY1,
tsx-33, supE44, falK2,
, rac
,
hisG4, rfbD1, mgl-51,
rpsL31, kdgK51, xyl-5,
mtl-1, argE3, and thi-1). The specific
strains were LM102 [AB1157, (F' traD36, proAB, LacIQZ
M15)], LM103
[AB1157uvrA6, (F' traD36, proAB,
LacIQZ
M15)], and LM119 [RPC501,
(F', traD36, proAB,
lacIQZ
M15)]. LM102, LM103,
and LM119 were constructed as previously described by Benamira and
Marnett (19). The E. coli strain JM105 was used as the host
bacterium for the replication of the M13MB102-1 genome on indicator plates.
M13mp7L2 was a generous gift from Christopher Lawrence (University of Rochester, Rochester, NY).
Construction of Adduct-modified M13MB102-1 Genomes-- Preparation of gapped-duplex DNA and ligation of adducted or unadducted 8-mers followed an adaptation of the method described by Benamira and Marnett (19). Double-stranded M13MB102-1 was linearized with SacII and BssHII followed by dialysis with a 12-fold excess of single-stranded M13MB102-1 in decreasing concentrations of formamide. The resultant gapped-duplex was resolved by 0.8% low melting point agarose gel electrophoresis. The band corresponding to gapped-duplex DNA was excised, and the DNA was recovered by GELase treatment. HOPdG and dG-containing 8-mers (100 pmol) were phosphorylated using T4 polynucleotide kinase and 1 mM ATP in the buffer supplied by New England BioLabs. For ligation, gapped-duplex DNA was added to each of the phosphorylated 8-mers, along with 400 units of T4 DNA ligase and 1 mM ATP. The reaction proceeded at 16 °C overnight in ligation buffer supplied by New England BioLabs. Reaction mixtures were brought up to 100 µl with H2O, and the DNA was purified by filtration through a modified polyvinylidene fluoride membrane from Millipore. Ligation products were purified on a 0.8% low melting point agarose gel and recovered using GELase enzyme.
Construction of M13mp7L2 Vectors Containing a Single HOPdG Adduct-- Single-stranded M13mp7L2 genomes containing a unique site-specific HOPdG were constructed, following a modification of the procedure described by Banerjee et al. (20). Briefly, single-stranded M13mp7L2 DNA was linearized with EcoRI for 2 h at 25 °C, utilizing the unique EcoRI site within the hairpin linker region. The linearized DNA was recircularized with a 48-mer oligonucleotide creating a circular single-stranded DNA containing an 8-base gap at the EcoRI site. The ends of the 48-mer oligonucleotide are complementary to the EcoRI site, and its internal sequence of 8 bases is complementary to the dG- and HOPdG-modified 8-mers. Recircularization was carried out in 20 mM MOPS/2 mM MgCl2/50 mM NaCl buffer (pH 7.4), by heating at 90 °C for 3 min and allowing the sample to cool slowly to room temperature overnight. A 100-fold molar excess of modified or unmodified 8-mer was ligated into the gapped vector using 400 units of T4 DNA ligase and 1 mM ATP at 16 °C overnight. The 48-mer scaffold was removed by addition of a 10-fold molar excess of anti-48-mer (the complement of the scaffold sequence) to the ligation mixture, followed by heating of the sample at 90 °C for 3 min and subsequent dilution with cold 1 mM Tris-HCl (pH 7.5), 0.1 mM EDTA. Samples were used immediately for transformation.
Mutagenesis Experiments--
Transformation of cells and
determination of mutation frequency were performed as described with
slight modifications (21). Briefly, bacteria were grown to logarithmic
phase and irradiated with UV light to induce the SOS response. The UV
dose was determined by irradiating cells at increasing times from 0 to
3 min and then plating dilutions of the irradiated cells on
Luria-Bertani plates. The optimal UV dose corresponded to roughly a
10% survival rate of the cells compared with no exposure. After
irradiation, the cells were made competent for transformation as
described previously (21). Competent cells (20 µl) were transformed
with 100 ng of single-stranded M13mp7L2 or 30 ng of double-stranded
M13MB102-1 using a BTX TransPorter Plus electroporation system.
Immediately following transformation, 1 ml of SOC medium (20 g/liter
bacto-tryptone/5 g/liter bacto-yeast extract/20 mM
glucose/2.5 mM KCl/10 mM MgCl2/9 mM NaCl) was mixed with the cells, and the bacteria were
plated on Luria-Bertani plates and incubated at 37 °C overnight
(22). To determine mutation frequencies for M13MB102-1, the entire
phage population was eluted from primary transformation plates,
diluted, and replated with JM105 on X-gal/IPTG indicator plates to give ~500 plaques per plate (23). In M13mp7L2 experiments, the primary transformation plates were used for mutation analysis. The plaques on
the plates were then lifted onto nylon membranes and probed for base
pair substitutions by differential hybridization with 13-mer probes for
the M13MB102-1 experiments (4) or 14-mer probes
(ATTCGGAXACCCAC, where X = A, C, G, or T)
for the M13mp7L2 experiments. Membranes from modified and unmodified
phage plates were split into four dishes, with each dish containing one
of the four hybridization probes.
Frameshift mutations induced by HOPdG in M13MB102-1 were detected by phenotypic screening with X-gal/IPTG during the secondary plating. M13MB102-1 harbors a single deletion at position 6284 of the lacZ gene, so frameshift mutations that restore the reading frame are detected as blue plaques against a background of colorless plaques.
In Vitro DNA Lesion Bypass Assays--
DNA polymerase assays
were performed essentially as described (24). Primers were
5'-radiolabeled using T4 polynucleotide kinase and
[-32P]ATP and purified with Bio-spin 6 columns
(Bio-Rad). Substrates were generated by mixing modified or unmodified
31-mer template with 32P-labeled primer in a 5:1
template:primer molar ratio in the presence of buffer (20 mM MOPS, pH 7.4, 2 mM MgCl2, and 50 mM NaCl) and incubating at 90 °C for 2 min, followed by
slow cooling to room temperature. Polymerase reaction mixtures (10 µl) contained 5 nM of the 32P-labeled DNA
template:primer substrate in 50 mM MOPS (pH 7.4), 10 mM MgCl2, 2 mg/ml bovine serum albumin, 5 mM dithiothreitol, with 100 µM dNTPs (dATP,
dCTP, dGTP, and dTTP individually or together as indicated) and the
indicated amounts of Klenow fragment (exo
). The reactions
were carried out for 30 min at 30 °C for the running-start primer
and 37 °C for the standing-start primer and were then quenched by
the addition of 10 mM EDTA in 90% formamide. The products
of the reaction were resolved on a 20% polyacrylamide gel using the
Ultrapure Sequagel sequencing system (National Diagnostics, Atlanta,
GA). The positions of the bands were visualized by autoradiography and
imaged with PhosphorImager analysis (Molecular Dynamics, Sunnyvale CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenicity of HOPdG-modified Double-stranded
Vectors--
Construction of vectors containing HOPdG followed the
basic procedure described by Benamira and Marnett (19). The M13MB102-1 vector was used during this preparation. M13MB102-1 contains a single
deletion at position 6284 in the lacZ gene of the
M13MB102 genome. Frameshift mutations that restore the reading frame of lacZ can be detected by phenotypic screening in the presence
of IPTG and X-gal. Double-stranded M13MB102-1 DNA was linearized with
BssHII and SacII, then dialyzed with an excess of
(+)-single-stranded phage DNA to create a duplex with an 8-nucleotide
gap in the (
) strand. Phosphorylated 8-mer oligonucleotides
containing HOPdG or dG were ligated into the gap at a position
corresponding to position 6256 in the M13 genome. The resultant closed
circular duplexes were separated from incompletely ligated duplexes in ethidium bromide-containing agarose gels. The modified genomes were
then transformed into E. coli strains that were either
wild-type or deficient in DNA repair. The transformed cells were plated to produce a lawn of progeny phage. The plaques were eluted, and an
aliquot of the stock was replated to yield ~500 plaques per plate.
Plaque DNA from the secondary plating was lifted onto nylon membranes
and probed by differential hybridization with radiolabeled probes
specific for each type of base pair substitution. Frameshift mutations
were detected by phenotypic screening using IPTG/X-gal during the
secondary plating. HOPdG did not increase the frequency of frameshift
mutations compared with unadducted genomes in any of the strains tested.
Table I lists the outcomes of in vivo replication when HOPdG-containing genomes were replicated in three separate E. coli strains. All strains were UV-irradiated before transformation to increase sensitivity to mutation by inducing the SOS response. No base pair substitutions were detected following replication of HOPdG-containing genomes in any of the strains with or without SOS induction. LM102 is wild-type for repair, whereas LM103 contains a uvrA6 mutation that eliminates nucleotide excision repair. No increase in mutation frequency was detected when the HOPdG-modified vectors were replicated in the LM103 strain. LM119 contains deletions in two major apurinic/apyrimidinic endonucleases, exonuclease III and endonuclease IV. Apurinic/apyrimidinic endonucleases function to cleave the phosphodiester backbone at abasic sites, and their action is associated with the base excision repair pathway. If base excision repair is involved in the removal of HOPdG, a deficiency in this repair pathway should result in an increase in the overall mutation frequency compared with repair-proficient cells. Replication of HOPdG-modified vectors in LM119 did not result in the occurrence of base pair substitutions.
|
Mutagenicity of Modified Single-stranded Vectors-- The use of duplex vectors may mask the mutagenicity of lesions because of DNA repair or strand bias during replication. Therefore, we constructed single-stranded vectors containing HOPdG at a defined site to eliminate these possibilities. Vector molecules were constructed by the method described by Banerjee et al. (20). Single-stranded M13mp7L2 was digested with EcoRI, utilizing a unique site within a short hairpin region formed in the single-stranded DNA. The vector was then recircularized by annealing with a scaffold oligonucleotide. The ends of the scaffold are complementary to the ends of the cut vector, but the scaffold leaves an eight-nucleotide gap within the duplex region. 8-mer oligonucleotides containing HOPdG or dG, in the same sequence context used during the construction of M13MB102-1 vectors, were ligated into the gap. The scaffold was removed by denaturation in the presence of an excess of its complement. The adduct-modified vector was transformed by electroporation into SOS-induced E. coli, and the transformed cells were plated to produce a lawn of progeny plaques. The plaques were lifted onto nylon membranes and assayed for mutations by differential hybridization with oligonucleotide probes specific for base pair substitutions. No mutations were observed in LM102 cells, complementing the results observed in the duplex vector studies (Table II).
|
In Vitro Lesion Bypass with Purified Polymerase--
The results
of the in vivo experiments suggest that HOPdG is not highly
mutagenic and is not a strong block to replication. Therefore, we
tested the ability of HOPdG to block DNA replication, using an in
vitro lesion bypass assay. Extension of a 12-mer running-start primer annealed with a 31-mer template containing HOPdG at the sixteenth position from the 3'-end was compared with an unmodified template. The template:primer substrate was extended by the Klenow fragment of DNA polymerase I (exo). The newly
synthesized products of this reaction were separated on a 20%
polyacrylamide gel and imaged using PhosphorImager analysis. Extension
of the primer opposite an unadducted strand by Klenow fragment in the
presence of all four dNTPs completely extended to the full-length
product. However, full-length extension of template:primers containing
HOPdG was substantially inhibited. Blockage of extension by Klenow
fragment occurred opposite to the thymidine immediately preceding HOPdG
(Fig. 2). At increased concentrations of
polymerase (molar ratio of enzyme to substrate greater than 1:1), a
second product was observed, corresponding to an arrest of replication
opposite HOPdG. Bypass of HOPdG to produce full-length extension
products was observed at all enzyme concentrations tested for Klenow
fragment (exo
), although the extent of bypass was
modest.
|
Primer extension studies were performed in the presence of individual
dNTPs using a 13-mer standing-start primer. Klenow fragment (exo) preferentially incorporated dAMP and
dGMP residues opposite HOPdG (dGMP > dAMP), although some
incorporation of dCTP was also observed (Fig.
3). Thus, in contrast to in
vivo replication, in vitro replication with Klenow
fragment reveals that HOPdG is able to strongly block replication and
to miscode for insertion of purines opposite to it.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we investigated both the mutagenic potential and
repair of an HOPdG adduct site-specifically positioned in double-stranded and single-stranded M13 genomes and replicated in
E. coli. Our results show that replication of
double-stranded vectors containing HOPdG does not induce base pair
substitutions or frameshift mutations at the level of sensitivity of
the assay (~103). The same results were obtained in
bacterial strains that are either wild-type for repair or deficient in
nucleotide excision repair or base excision repair. This observation
does not result from a masking effect due to the double-stranded nature
of the vector, because HOPdG also was not mutagenic when incorporated into single-stranded vectors. Thus, even though the entire base pairing
region of guanine is blocked, DNA polymerases still appear to insert C
opposite HOPdG in vivo.
The low mutagenicity of HOPdG is quite different from that of other six-membered exocyclic guanine derivatives when compared in an identical sequence context. PdG and M1G induce base pair substitutions and frameshift mutations in vitro and in vivo (2, 4). HOPdG, M1G, and PdG do not induce frameshift mutations in the sequence used in the present study, but PdG and M1G do induce frameshifts in a sequence containing reiterated CGs. The structural difference between PdG and HOPdG is the presence or absence of a hydroxyl group, whereas M1G and HOPdG also differ by the pi character of the exocyclic ring of M1G (Fig. 1). An analogy may be drawn to the study of Langouet and coworkers (25), which demonstrated that ethano-dG and etheno-dG, the five-membered homologues of PdG and M1G, are mutagenic in E. coli, whereas HO-ethano-dG, the homologue of HOPdG, is only weakly mutagenic.
The lack of mutations induced by HOPdG is not the result of it being a
strong block to in vivo replication or to its removal by
repair systems, as demonstrated by the use of single-stranded vectors
in mutagenesis assays. One possible explanation for HOPdG's nonmutagenic nature is that the structure of HOPdG may exist in multiple forms, where one form is miscoding but the other not (Fig.
4). A related interconversion has been
observed for M1G, which exists in a hydrolytically
ring-opened form (N2-(3-oxopropenyl)-dG) when it
is opposite dC residues but in the ring-closed form when it is opposite
dT residues (Fig. 4). The ring-opened form appears to be substantially
less mutagenic than the ring-closed form. One can postulate the
existence of an analogous ring-opened form for HOPdG in which the
ring-opened form, N2-(3-oxopropanyl)-dG, would
be expected to be substantially less mutagenic. In fact, a very recent
study by de los Santos et al. (26) indicates that HOPdG
exists entirely in the ring-opened form in duplex DNA and that the
ring-opened form hydrogen bonds effectively to dC residues. This type
of interconversion of opened and closed forms of exocyclic adducts may
account for the low mutagenicity of HO-ethano-dG as speculated by
Langouet et al. (25).
|
The relevance of our results to the mutagenicity and tumor-initiating
activity of acrolein is supported by recent studies by Yang and
coworkers (27), who reported that HOPdG was not mutagenic in E. coli. It seems unlikely that HOPdG is the principal adduct
responsible for acrolein mutagenicity. This suggests that the mutagenic
effects are mediated by other acrolein-DNA adducts. Isomeric 6-HOPdG
isomers are formed on reaction of acrolein with DNA in vitro
and have been detected in human tissue (12, 13, 15). Even if 6-HOPdG
ring-opens in duplex DNA, it will still contain a 3-oxopropanyl group
on N1 of dG, which should alter translesion synthesis. In addition,
there are other adducts that may contribute to the genotoxicity of
acrolein. Recently, Wang et al. (28) reported that the
hydrolysis product of crotonaldehyde--hydroxybutyraldehyde adds to
the exocyclic amino group of deoxyguanosine to form unstable imine
adducts. The imine adducts are present in concentrations 50-fold higher
than those of the 6-methyl-8-hydroxypropanodeoxyguanosine adduct
generated by reaction of deoxyguanosine with crotonaldehyde. It is
likely that related N2-imino adducts are formed
by reaction of deoxyguanosine residues with hydrated acrolein
(i.e. 3-hydroxypropionaldehyde). It will be interesting to
evaluate the mutagenicity of these adducts using site-specific
procedures analogous to those described in the present work. However,
the chemical instability of these novel exocyclic and
N2 adducts may make this a formidable
experimental challenge.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Rouzer for a critical reading of the manuscript. We are very grateful to M. Moriya and C. de los Santos for sharing the results of their experiments with HOPdG prior to publication.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Research Grants CA47479, CA87819, ES07781, and ES00267 and Center Grant CA68485 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Training Grant ES07028 from the National Institutes of Health.
To whom correspondence should be addressed: Dept. of
Biochemistry, Vanderbilt University School of Medicine, 23rd Ave. at Pierce, Nashville, TN 37232. Tel.: 615-343-7329; Fax: 615-343-7534; E-mail: marnett@toxicology.mc.vanderbilt.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008900200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
M1G, pyrimido[1,2-]purin-10(3H)-one;
PdG, 1,N2-propano-2'-deoxyguanosine;
HOPdG, 8-hydroxypropanodeoxyguanosine;
MOPS, 4-morpholinepropanesulfonic acid;
IPTG, isopropyl-
-D-thiogalactoside;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Singer, B., and Bartsch, H. (1999) Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis , International Agency for Research on Cancer, Lyon, France |
2. |
Fink, S. P.,
Reddy, G. R.,
and Marnett, L. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8652-8657 |
3. |
Moriya, M.,
Zhang, W.,
Johnson, F.,
and Grollman, A. P.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11899-11903 |
4. |
Burcham, P. C.,
and Marnett, L. J.
(1994)
J. Biol. Chem.
269,
28844-28850 |
5. | Beauchamp, R. O., Jr., Andjelkovich, D. A., Kligerman, A. D., Morgan, K. T., and d'A Heck, H. (1985) CRC Crit. Rev. Toxicol. 14, 309-380 |
6. | Marnett, L. J. (1988) in Air Pollution, the Automobile, and Public Health (Watson, A. Y. , Bates, R. R. , and Kennedy, D., eds) , pp. 579-603, Health Effects Institute, National Academy Press, Washington, DC |
7. |
Uchida, K.,
Kanematsu, M.,
Morimitsu, Y.,
Osawa, T.,
Noguchi, N.,
and Niki, E.
(1998)
J. Biol. Chem.
273,
16058-16066 |
8. | Brock, N., Stekar, J., Pohl, J., Niemeyer, U., and Scheffler, G. (1979) Arzneim.-Forsch. 29, 659-661[Medline] [Order article via Infotrieve] |
9. | Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Mutat. Res. 148, 25-34[Medline] [Order article via Infotrieve] |
10. | Carson, B. L., Beall, C. M., Ellis, H. V., Baker, L. H., and Herndon, B. L. (1981) Gov. Rep. Announce. (U. S. Index) 12 |
11. | Cohen, S. M., Garland, E. M., St. John, M., Okamura, T., and Smith, R. A. (1992) Cancer Res. 52, 3577-3581[Abstract] |
12. | Chung, F.-L., Young, R., and Hecht, S. S. (1984) Cancer Res. 44, 990-995[Abstract] |
13. | Chung, F.-L., Roy, K. R., and Hecht, S. S. (1988) J. Organic. Chem. 53, 14-17 |
14. | Foiles, P. G., Akerkar, S. A., and Chung, F.-L. (1989) Carcinogenesis 10, 87-90[Abstract] |
15. | Nath, R. G., and Chung, F.-L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7491-7495[Abstract] |
16. | Chung, F. L., Nath, R. G., Nagao, M., Nishikawa, A., Zhou, G. D., and Randerath, K. (1999) Mutat. Res. 424, 71-81[CrossRef][Medline] [Order article via Infotrieve] |
17. | Khullar, S., Varaprasad, C. V., and Johnson, F. (1999) J. Med. Chem. 42, 947-950[CrossRef][Medline] [Order article via Infotrieve] |
18. | Nechev, L. V., Harris, C. M., and Harris, T. M. (2000) Chem. Res. Toxicol. 13, 421-429[CrossRef][Medline] [Order article via Infotrieve] |
19. | Benamira, M., and Marnett, L. J. (1993) Chem. Res. Toxicol. 6, 317-327[Medline] [Order article via Infotrieve] |
20. | Banerjee, S. K., Borden, A., Christensen, R. B., LeClerc, J. E., and Lawrence, C. W. (1990) J. Bacteriol. 172, 2105-2112[Medline] [Order article via Infotrieve] |
21. | Fink, S. P., Reddy, G. R., and Marnett, L. J. (1996) Chem. Res. Toxicol. 9, 277-283[CrossRef][Medline] [Order article via Infotrieve] |
22. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd. Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
23. |
Benamira, M.,
Singh, U. S.,
and Marnett, L. J.
(1992)
J. Biol. Chem.
267,
22392-22400 |
24. |
Hashim, M. F.,
and Marnett, L. J.
(1996)
J. Biol. Chem.
271,
9160-9165 |
25. | Langouet, S., Mican, A. N., Muller, M., Fink, S. P., Marnett, L. J., Muhle, S. A., and Guengerich, F. P. (1998) Biochemistry 37, 5184-5193[CrossRef][Medline] [Order article via Infotrieve] |
26. |
de los Santos, C.,
Zaliznyak, T.,
and Johnson, F.
(2001)
J. Biol. Chem.
276,
9077-9082 |
27. |
Yang, I.-Y.,
Hossain, M.,
Miller, H.,
Khullar, S.,
Johnson, F.,
Grollman, A.,
and Moriya, M.
(2001)
J. Biol. Chem.
276,
9071-9076 |
28. | Wang, M., McIntee, E. J., Cheng, G., Shi, Y., Villalta, P. W., and Hecht, S. S. (2000) Chem. Res. Toxicol. 13, 1065-1074[CrossRef][Medline] [Order article via Infotrieve] |