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Carcinogenesis |
Molecular and Cellular Pharmacology Program [R. L. L.], Molecular and Cellular Biology Program [M. H.], and Laboratory for Chemical Biology, Department of Pharmacological Sciences [I-Y. Y., A. P. G., M. M.], State University of New York at Stony Brook, Stony Brook, New York 11794-8651, and American Health Foundation [G. A. P.], Valhalla, New York 10595
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dA) in human
cells, a novel site-specific mutagenesis approach was developed, in
which a single DNA adduct was uniquely placed in either strand of a
shuttle plasmid vector. The analysis of progeny plasmid derived from
the modified strand shows that dA, when incorporated into the
position of the second A of 5'-CAA (codon 61 of the ras
gene), is mutagenic in human cells, inducing A
T, AG, and AC
mutations. The efficient induction of AT transversions in
experiments using modified double- and single-stranded DNA substrates
supports the hypothesis that A:TT:A transversions in human and
animal tumors induced by vinyl compounds reflect misinsertion of dAMP
opposite this adduct. Mutagenic events were similar when the adduct was
incorporated into either the leading or the lagging strand. dA was
more mutagenic than 8-oxodeoxyguanosine, which induced targeted GT
transversions in HeLa cells. In Escherichia coli, dA
did not significantly miscode (<0.27%) even in the presence of
induced SOS functions. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dA,3
a member of a family of exocyclic DNA adducts, is produced by
endogenous and exogenous agents and has been shown to be mutagenic
in vitro (2
, 3)
and in vivo
(4
, 5)
. dA forms when reactive metabolites of vinyl
compounds, such as VC, vinyl carbamate, and urethane (reviewed in Ref.
6
), and certain 
,ß-unsaturated aldehydes generated by
lipid peroxidation (reviewed in Refs. 7
and
8
) react with DNA. dA was detected in human liver DNA
at the level of 
20 adducts per 109 dA
(9
, 10)
. The level of dA in DNA is increased by
enhanced lipid peroxidation (10
, 11)
.
1,N6-Ethenoadenine was detected in the
urine of untreated rats at the level of 21.6 pmol/ml (12)
.
In human tumors associated with exposure to VC and in animal tumors
induced by administration of vinyl compounds, A:TT:A transversions
were observed in the p53 tumor suppressor gene
(13, 14, 15)
and the ras oncogenes (codon 61; Refs.
16, 17, 18
). In previous studies using ss plasmid DNA, dA
was strongly mutagenic in simian kidney cells (5)
.
dAdG and dAdT base changes accounted for 63 and 6%,
respectively, of translesional events. However, the strong dominance of
dAdG in this experiment is inconsistent with the frequently
observed A:TT:A transversions in those tumors.
Among mutations at the second base of the c-Ha-ras codon 61
(CAA), AT and AG mutations show similar transforming activities
(19
, 20) . A:TT:A mutations in codons 179, 249, and 255
of the p53 gene were observed in human liver angiosarcomas
associated with exposure to VC (13
, 14)
. Analysis of a
database for human p53
mutations4
reveals 17 and 52 examples of AT and AG mutations, respectively,
at codon 179 (CAT), and 21 and 13 AT and AG mutations,
respectively, at codon 249 (AGG). Taken together, these findings
suggest that phenotypic selection does not account for the A:TT:A
mutations observed at these sites in the p53 and
ras genes in vinyl compound-associated tumors.
In this paper, we describe an experimental system that can be used with
dsDNA and ssDNA substrates to study mutagenic events in human cells.
With it, we found that dA directs the incorporation of dAMP opposite
the adduct when embedded in the sequence 5'-CAA, in which the
modified base corresponds to the second base of codon 61 of the
ras oncogene.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dA-containing oligonucleotides were obtained from Oligos Etc.
(Wilsonville, OR). An oligonucleotide containing 8-oxo dG was provided
by F. Johnson (State University of New York at Stony Brook, Stony
Brook, NY). Unmodified and modified oligonucleotides were purified by
electrophoresis in a denaturing 20% polyacrylamide gel, followed by
purification by high-performance liquid chromatography and then
subjected to the analysis for base composition as described earlier
(5)
. To examine the stability of dA, the
oligonucleotide was incubated in solutions used during construction and
subjected to electrospray mass spectrometry. This analysis indicated
that dA is stable under the construction conditions.
Cell Lines and Bacteria.
HeLa (cervical cancer), HCT116 (mismatch repair-defective colon
cancer), and 293 (embryonal kidney epithelium) cells were obtained from
American Type Culture Collection (Rockville, MD). They were free of
Mycoplasma. Cells were grown in DMEM supplemented with 10%
fetal bovine serum and antibiotics. Electrocompetent Escherichia
coli DH10B was purchased from Life Technologies, Inc.
(Gaithersburg, MD). MM1991 (as AB1157, but
mutS201::Tn10,
endA7::cm) was constructed by P1
transduction.
Construction of Vectors.
Three modifications were introduced into our pMS2 vector (Fig. 1
; Ref. 21
): (a) we introduced the
human BK virus origin and BK large T antigen gene into the unique
HindIII site of pMS2. These elements allowed the vector to
replicate in human cells. For this modification, the BK shuttle vector
(National Biosciences, Plymouth, MN) was digested with
PvuII, excising a 3.3-kb fragment containing the two
elements. This fragment was ligated to the HindIII site of
pMS2 in the orientation shown in Fig. 1
, creating pBK20 (8.4 kb);
(b) we transferred HP (HP in Fig. 1
), the site
for incorporation of a DNA adduct, downstream of the neo
gene so that the DNA adduct was remote from the viral origins of
replication. The transfer assured that TLS would be catalyzed during
the elongation and not during the initiation stage. For this
modification, pBK20 was digested with XhoI, and two
fragments were isolated. The larger fragment (7.2 kb) was circularized
by self-ligation creating pBK30, and the 1.2-kb smaller fragment,
containing the neo gene, was ligated to EcoRI
linkers at both ends. This neo gene fragment was then
introduced into the unique EcoRI site located immediately
upstream of HP (Fig. 1
) in pBK30, creating pSBK; and (c)
pSBK also contains the base change of GATCAT at codon 402 of the
large T antigen gene. This change substituted His for Asp, disrupting
the interaction of T antigen with p53 protein (22
, 23)
.
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Construction of DNA Containing a Single DNA Adduct.
Fig. 2
shows the construction schematically. ds pSBK was digested with
EcoRV to remove a 33-bp fragment from the HP region (Fig. 2
,
step 1). The digested vector was ligated to a blunt-ended
13-mer duplex [5'-d(AGGTACGTAGGAG)/3'-d(TCCATGCATCCTC)] containing a
SnaBI site (5'-TACGTA; Fig. 2
, step 2). Two
constructs (pSBKG and pSBKD) were isolated, each having a single insert
in the opposite orientation. For the leading strand construct, 60 pmol
(162 µg) of ss pSBKD, prepared from JM109 harboring this plasmid,
with the aid of the helper phage M13KO7 (Life Technologies, Inc.), was
mixed with 20 pmol (108 µg) of EcoRV-digested ds pSBK.
Gapped DNA was prepared according to the method of Horiuchi and Zinder
(24)
. Annealing between circular ss pSBKD and its
complementary strand, derived from EcoRV-digested ds pSBK,
formed gapped DNA with a 13- nucleotide gap (Fig. 2
, step
4). The formation of gapped DNA was confirmed by electrophoresis
in a 0.8% agarose gel (Fig. 3
). The DNA solution was desalted using a Centricon 30 Microconcentrator
(Amicon, Beverly, MA). One hundred pmol of gel-purified control or
adducted 13-mer [5'-d(CTCCTCXATACCT), where X is dA or dA], were
phosphorylated at the 5' termini by T4 polynucleotide kinase and ATP,
and ligated to the gapped DNA by T4 DNA ligase (100 units/µg DNA) at
10°C for 24 h (Fig. 2
, step 5). After desalting in a
Centricon 30, the ligation mixture was treated with SnaBI
and EcoRV to remove undesired products. The desired
construct is resistant to these restriction enzymes. The formation of
cc dsDNA was confirmed by agarose gel electrophoresis (Fig. 3
), and the
product was purified by ethidium bromide/CsCl density-gradient
centrifugation (25)
. After the removal of ethidium bromide
and CsCl, DNA was concentrated and washed with
H2O in a Centricon 30. The concentration of cc
dsDNA construct was determined spectrophotometrically. The final yield
ranged from 2 to 10% based on the initial amount of ds pSBK. Gapped
DNA for lagging constructs were prepared similarly, using
EcoRV-digested ss pSBK and ds pSBKG (Fig. 2
). The
construction of ssDNA bearing a single adduct has been described
previously (5)
.
|
|
. This method allowed positive detection of sequences complementary to
the probe because even a single base mismatch inhibits hybridization
between probe and target DNA. Plasmids containing dA, dC, dG, or dT at
the position of the adduct were included on the filter as
positive controls. None of the probes cross-hybridized. Plasmids that
contained mutations other than a targeted mutation did not hybridize to
any of the AA, AT, AG, AC, or AD probes (Fig. 4
). Their sequence
changes were determined by DNA sequencing. Thus, all types of mutations
were detected and determined by this technique.
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dA and electroporated using the E.
coli-Pulser (Bio-Rad). After the addition of 950 µl of 2x YT,
bacteria were cultured for 40 min. A small volume (1–10 µl) of
transformation mixture was plated on 1x YT-ampicillin (100 µg/ml)
plates to determine the number of transformants. After incubating the
remaining transformation mixture for 20 min, the culture was added to
19 ml of 2x YT-ampicillin and cultured overnight. Plasmid DNA was
prepared from an overnight culture by alkaline-lysis, was digested with
SnaBI, and was used to transform E. coli DH10B.
Transformants were analyzed for mutations as described above. |
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP at its 5' end and was used to
construct heteroduplex DNA. Upper and lower bands were collected
separately and digested with XhoI. As shown in Fig. 5
, two XhoI sites were located near the ligation sites. When
the oligonucleotide was ligated to gapped DNA at its 5' and 3' ends,
XhoI treatment generated a labeled 61-mer, whereas partially
ligated products yielded a labeled 40-mer and 33-mer, respectively,
when ligated to DNA at the 5' or 3' end of the insert. Only a 61-mer
was observed for DNA recovered from the lower band (Lane 2),
whereas 34-mer, 40-mer, and 61-mer were observed for DNA recovered from
the upper band (Lane 1). These results established that the
lower band contained fully ligated products.
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), whereas progeny derived from TLS hybridized to the L
and R probes and to one of the A probes (Fig. 4
). Progeny with
deletions in the region of analysis did not hybridize to any of the
probes. The analysis of progeny plasmid revealed that the ratio of
progeny derived from the two strands was close to 1:1 (55
versus 45%) for the control construct (Table 1)
which suggests that each strand replicated equally. In contrast, the
fraction of progeny derived from the unmodified strand was much larger
than that from the modified strand when the modified construct was used
(88 versus 9%). This suggests that the dA adduct blocked
DNA synthesis. Another possibility was excision repair of dA.
Removal of this adduct and the 5' and 3' flanking mismatched bases,
followed by the filling of the gap, created a SnaBI site in
the modified strand. There was a 2-fold increase in the number of
progeny containing large deletions in the modified construct, compared
with that observed in the control (Table 1)
.
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shows frequencies of progeny that contain deletions obtained after
SnaBI enrichment. For both leading and lagging control
constructs, deletion frequency was highest in HCT116 and lowest in
HeLa. Deletions ranged from several hundred to 2000 bp. When
modified constructs were used, the fractions of deletion mutants
increased 4-fold for HeLa and 293 cells and 2-fold for HCT116
(Table 2)
. These increased frequencies resulted from a relative
increase of a fraction of deletion mutants in progeny after
SnaBI digestion. When data in Table 1
were recalculated,
ignoring progeny derived from the SnaBI strand, the
frequencies of deletions are 2.7% (6 of 222) for the control
and 21% (12 of 57) for the modified construct. The results of
experiments shown in Table 2
indicated that TLS analysis in HeLa cells
was the least labor-intensive. Therefore, we used HeLa cells as the
primary host for TLS analysis.
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Mutagenesis of dA in Human Cells.
To determine the number of G418-resistant cells per transfection, 5%
of the transfected cells were plated separately and maintained in
complete medium containing G418 for 12–14 days. G418-resistant
colonies were counted after staining with Giemsa. The number of
transfected cells ranged from 2000 to 6000 per µg of construct for
the experiments shown in Table 3
. The number of G418-resistant colonies does not represent the degree of
TLS but shows simply the transfection efficiency.
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. This analysis showed 14 and 10% targeted miscoding frequencies
in HeLa cells for the leading and lagging strand constructs,
respectively. This indicated that dTMP was preferentially inserted
opposite dA at frequencies of 86 to 90% despite the lack of normal
hydrogen bonding expected by the presence of the exocyclic ring. In
another experiment, in which a leading strand construct was purified
twice by ultracentrifugation, the frequency of miscoding was 14%. In
an experiment with HCT116, the fraction of progeny derived from TLS was
small as expected; mutation frequency was 7% for the leading strand
construct (Table 3)
. Although the incidence varied, all types of base
substitution mutations were observed. When the identical modified
oligonucleotide was inserted into ssDNA and replicated in HeLa cells,
results were similar to those observed for dsDNA (Table 3)
.
To validate this system, another DNA adduct, 8-oxo dG, was incorporated
into the leading strand. This adduct induced GT transversions in
HeLa cells at a frequency of 1%. This result was consistent with that
of a previous study in COS cells (21)
and suggests that
the mutational specificity observed for dA reflects its intrinsic
miscoding properties in human cells.
Three types of other sequence changes were observed when modified
constructs were used (Table 4)
. A common feature of these mutations is a T:CT:A change 5' to the
adduct site. Many of those were accompanied by an additional targeted
base change of G:AG:C, i.e.,
5'-GT/3'-AC5'-GT/3'-CA ("mutant b" in Table 4
). Mutants with
this sequence change were observed reproducibly and accounted for 80%
(24 of 30) of mutants with "other sequence changes" (Table 3)
.
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dA in E. coli.dA was used to transform
MM1991. Progeny plasmid was prepared from a transformation mixture
after overnight incubation in the presence of ampicillin and was used
to transform E. coli DH10B. An analysis of DH10B
transformants did not show any targeted point mutations; miscoding
frequencies were <0.27% in the presence or absence of induced SOS
functions. One plasmid obtained in the presence of induced SOS
functions had the same sequence change as mutant b. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While developing this approach, we found that plasmid that is maintained in human cells is associated with a high incidence of large deletions. The cause of these deletion mutations is not clear. They are not attributable to nicks in the constructs, inasmuch as we isolated cc dsDNA by ultracentrifugation, and in HeLa cells, even supercoiled dsDNA plasmid yielded deletion mutations at a similar frequency. Furthermore, the incidence of large deletions depends on host cells. It is not known what contributes to the difference in the spontaneous frequency of large deletions between transformed human cell lines. Among the three cell lines tested, plasmid was most unstable in HCT116. This may be ascribed to its mutator phenotype attributable to mismatch repair deficiency. For our purposes, HeLa cells proved to be the best host, showing the lowest incidence of deletions.
Our results show that dA is substantially mutagenic in human
cells. dA did not miscode in E. coli when embedded in the
same sequence. This confirms previous results obtained with ssDNA
(5)
. Basu et al. (29)
reported a
limited number of AG transitions when dsDNA containing dA was
replicated in vitro by HeLa cell extracts. Recently,
E. coli UmuD'2C complex (DNA
damage-inducible proteins involved in error-prone TLS pathway) was
discovered to have an error-prone DNA polymerase activity and
designated DNA polymerase V (30)
. In E. coli,
the dA adduct inhibits DNA synthesis, and the induction of
UmuD' and UmuC proteins increases the level of
TLS.5
Our results in E. coli (Table 3)
show that dTMP is the
preferred nucleotide during TLS, which suggests that pol V may
catalyze error-free DNA synthesis across this adduct.
Which DNA polymerase is responsible for the mutations observed in human
cells? Human cells have at least two additional DNA polymerases,
pol 
and pol 
(31, 32, 33)
. Both polymerases
catalyze TLS across cyclobutane pyrimidine dimers. Whereas pol
catalyzes error-free TLS, pol conducts error-prone TLS. Studies
with XP variant cells, which lack pol , could be used to determine
the role of pol in mutagenesis induced by this adduct.
With regard to miscoding frequency and specificity, results obtained in
HeLa and HCT116 cells are quite different from those obtained using
simian kidney cells (COS; 5
). In our COS cell experiments,
miscoding frequency reached 70% with dAdG mutations being
strongly dominant. There are several differences in the design of these
two studies, including ss versus ds vector, sequence
context, location of the DNA adduct relative to the replication origin,
and host cells. One or several of these factors could have contributed
to the differences observed. Because the experiment using ssDNA in HeLa
cells yielded a miscoding frequency similar to that using dsDNA, the
difference between ssDNA and dsDNA does not account for the
discrepancy.
The analysis of tumors in humans and animals exposed to VC or vinyl
carbamate showed A:TT:A transversions in the p53 and
ras genes (13, 14, 15, 16, 17, 18)
. The results reported here
show that dA efficiently induces this type of transversion in human
cells. There are two possible mechanisms by which dA might induce
dAdT transversions. One possibility is the incorporation of dAMP
opposite the adduct. The other is via the formation of an abasic site.
dA is known to be removed by human 3-methyladenine DNA glycosylase
(34)
, which creates an abasic site that is then repaired
by apurinic/apyrimidinic endonuclease (35)
. If TLS occurs
at the abasic site, AT transversions will be induced as the
consequence of dAMP insertion opposite the lesion (36
, 37)
. It is reported that an imbalance between the DNA
glycosylase activity and the endonuclease activity increases the
spontaneous mutation frequency (38)
. However, we believe
this mechanism to be unlikely because dA embedded in ssDNA shows
mutational events similar to those in dsDNA (Table 3)
. The DNA
glycosylase is not active on dA located in ssDNA (39)
.
Therefore, we conclude that the A:TT:A transversions observed in
human and animal tumors were induced by misreplication of dA. A
remaining question is why dAdG transitions were not observed
equally in those tumors. This type of base substitution at the sites
(described in the "Introduction") in the p53 and
ras genes are not silent as discussed in the
"Introduction," and dA induces this mutation at a frequency
similar to that for dAdT. In one tumorigenicity study with VC
(15)
, AT, AG, and AC mutations were observed in
the rat p53 gene, consistent with the results of our study.
Our results show that dA shows a similar miscoding frequency when
located in either strand. This contrasts with the finding in E.
coli that acetylaminofluorene-dG adduct showed a marked difference
in its mutagenic activity when located in the leading or lagging strand
(40)
.
dA is produced endogenously, and its level in DNA is high in
Wilson’s disease and primary hemochromatosis (10)
. These
metal storage disorders are associated with increased frequencies of
liver tumors (41, 42, 43)
. The finding of its endogenous
origin and increased formation following enhanced lipid peroxidation,
coupled with the demonstrated mutagenic activity in human cells,
implies that dA is a significant threat to genomic integrity.
In addition to targeted single mutations, other sequence changes were
observed in the adducted region (Tables 3
4)
. Whereas targeted
single mutations are derived from TLS because they are flanked by 5'-C
and 3'-A, the origin of the other sequence changes is not clear. These
sequence changes can be explained by untargeted single-point mutations
in either strand (Table 4)
. Because progeny plasmid was digested with
SnaBI, any change in the recognition sequence in the
complementary strand renders progeny resistant to the digestion.
Mutants b and c could arise, respectively, from CT and GT
mutations in 5'-CGT of the complementary strand (Table 4)
. Mutant d
could be derived from CA in 3'-AXC of the adducted strand. Among
these sequence changes, mutant b accounts for the majority. In addition
to the above possibility, this dominant sequence change can be
generated by template strand switching at the adduct site (Fig. 6
). If this mechanism operates, the blocked 3' end of the nascent strand
must be extended from a G:T mismatched terminus. It is reported that a
G:T mismatch is the easiest to be extended among various mismatches
(44)
. Additional studies are under way to distinguish
these possibilities.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA76163 (to M. M. and
PO1CA47995 (to A. P. G.), Pharmaceutical Research and
Manufacturers of America Foundation (to R. L. L.), and NIH Training
Grant T32-GM08468 (to M. H.). 
2 To whom requests for reprints should be
addressed, at Department of Pharmacological Sciences, State University
of New York at Stony Brook, Stony Brook, NY 11794-8651. Fax:
(631) 444-7641; E-mail: maki{at}pharm.sunysb.edu
3 The abbreviations used are: dA,
1,N6-ethenodeoxyadenosine; cc, closed
circular; ds, double-stranded; ss, single-stranded; TLS, translesion
DNA synthesis; VC, vinyl chloride; 8-oxo dG, 8-oxodeoxyguanosine; HP,
hairpin sequence; pol, polymerase.
4 Internet address:
http://perso.curie.fr/tsoussi.
5 I-Y. Yang, G. A. Pandya, and M. Moriya,
unpublished results.
Received 1/11/00. Accepted 5/26/00.
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I.-Y. Yang, M. Hossain, H. Miller, S. Khullar, F. Johnson, A. Grollman, and M. Moriya Responses to the Major Acrolein-derived Deoxyguanosine Adduct in Escherichia coli J. Biol. Chem., March 16, 2001; 276(12): 9071 - 9076. [Abstract] [Full Text] [PDF]
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E. Speina, J. M. Ciesla, J. Wojcik, M. Bajek, J. T. Kusmierek, and B. Tudek The Pyrimidine Ring-opened Derivative of 1,N6-Ethenoadenine Is Excised from DNA by the Escherichia coli Fpg and Nth Proteins J. Biol. Chem., June 8, 2001; 276(24): 21821 - 21827. [Abstract] [Full Text] [PDF]
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R. L. Levine, H. Miller, A. Grollman, E. Ohashi, H. Ohmori, C. Masutani, F. Hanaoka, and M. Moriya Translesion DNA Synthesis Catalyzed by Human Pol eta and Pol kappa across 1,N6-Ethenodeoxyadenosine J. Biol. Chem., May 25, 2001; 276(22): 18717 - 18721. [Abstract] [Full Text] [PDF]
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