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INTRODUCTION |
Somatic mutation of proto-oncogenes and tumor suppressor genes is
a key component in the initiation of cancer (1). Any DNA damage that
escapes repair could lead to mutations. However, replicative DNA
polymerases are blocked in a potentially lethal manner when they
encounter bulky adducts (2). Thus, they are unable to convert adducts
to mutations. Recently, a growing number of DNA polymerases capable of
conducting translesion DNA synthesis have been discovered (3-11);
these likely hold the key to mutagenesis and the initiation of cancer
induced by bulky adducts. One of these lesion-bypassing DNA
polymerases, human DNA polymerase eta (pol
)1 (12, 13) is a
member of the UmuC/DinB/Rev1/Rad30 superfamily (now called
the Y family (14)) of DNA polymerases. In humans it is a product of the
XPV skin cancer susceptibility gene; inactivation of pol
results in a variant form of xeroderma pigmentosum (12, 15). pol
incorporates mostly correctly at cis-syn T-T
cyclobutane dimers (16, 17),
N-(deoxyguanosin-8-yl)-acetylaminofluorene (18), and
cis-Pt G-G adducts (18, 19); small amounts of incorrect
nucleotides are also incorporated, mostly at the level seen with
undamaged DNA. With (6-4) T-T photoproducts, however, a
single G is preferentially incorporated (20).
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental
carcinogens (21). Bay-region diol epoxides, formed on an angular
benzo-ring, have been shown to be the ultimate carcinogenic metabolites
of the PAHs (21-25). Four optically active bay-region diol epoxide
isomers (enantiomers of a pair of diastereomers) are formed
metabolically from a given hydrocarbon. In the DE-1 ("syn")
diastereomer, the epoxide oxygen and benzylic 7-hydroxyl groups are
cis, whereas in the DE-2 ("anti") diastereomer these groups are
trans (see Fig. 1A below). For bay-region diol
epoxides, the DE-2 isomer with
R,S,S,R absolute
configuration exhibits by far the greatest carcinogenicity compared
with the other three optically active isomers, whereas for fjord-region
diol epoxides significant carcinogenic activity is not limited to this
isomer (26).
The exocyclic amino groups of purine bases (N2
of G and N6 of A) in DNA are the principal
targets of PAH diol epoxides (27). Cis and trans addition of the amino
group to the benzylic epoxide position of the diol epoxide produces
adducts that have either retained or inverted configuration,
respectively, at the point of attachment (C-10 for
benzo[a]pyrene) to the hydrocarbon moiety. Fig.
1B shows how cis addition at C-10 of
(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-BaP DE-2) results in retention of configuration at C-10
(10R 
10R) whereas trans addition results in
inversion of configuration (10R 10S).
The present study utilizes N2 dG adducts derived
from the carcinogenic (+)-BaP DE-2 and the weakly carcinogenic or
non-carcinogenic (26)
(
)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (()-BaP DE-2). A report (28) of pol activity at the trans (10S) dG adduct of (+)-BaP DE-2 indicated that A and T were
frequently misincorporated; however, only the 10S adduct was
investigated. In the present study we report that misincorporation of
purine nucleotides by pol commonly occurs opposite all four types of dG adducts (cis/trans and 10R/10S) formed by
carcinogenic (+)-BaP DE-2 and non-carcinogenic ()-BaP DE-2, with the
adducts placed in two different DNA sequence contexts (Fig.
1C).
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Fig. 1.
Adducts formed by BaP DE-2 isomers.
A, the two metabolically formed diastereomers of the
bay-region benzo[a]pyrene diol epoxide (BaP DE). In DE-1
the benzylic 7-hydroxyl group and epoxide oxygen are cis, and in DE-2
these groups are trans. Each diastereomer consists of a pair of
enantiomers. B, partial structures of the BaP DE-2
enantiomers,
(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene
((+)-BaP DE-2) and
()-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene
(()-BaP DE-2), are shown along with the ring-opened
products formed upon their reactions with the exocyclic
N2-amino group of dG (indicated as
NHR). Cis opened adducts retain configuration at C-10,
whereas trans adducts have inverted configuration. C, the
DNA 16-mers, contexts III(G) and IV(G), used as templates in the
present study. The adduct is indicated as G*.
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MATERIALS AND METHODS |
Oligonucleotides--
Oligonucleotides containing a single
specific cis or trans BaP DE-2 dG adduct placed on the fourth
nucleotide from the 5'-end in either sequence context III(G)
(5'-TTCG*AATCCTTCCCCC-3') or IV(G) (5'-GGGG*TTCCCGAGCGGC-3') were
synthesized as described previously (29, 30). The basis for selection
of these sequences has been described previously (30). Polyacrylamide
gel-purified, non-adducted oligomers were purchased from either
Lofstrand Laboratories Limited (Gaithersburg, MD) or Midland Certified
Reagent Co. (Midland, TX) and were purified further by
reverse-phase high performance liquid chromatography if necessary.
DNA Polymerase Reactions--
His-tagged recombinant DNA
polymerase was produced as described previously (18). Polymerase
reactions contained final concentrations of 40 mM Tris-HCl
(pH 8.0), 1 mM MgCl2, various concentrations of
the four dNTPs, 10 mM dithiothreitol, 250 µg/ml bovine
serum albumin, 60 mM KCl, 2.5% glycerol, 40 nM
5'-32P-labeled primer previously annealed to 60 nM 16-mer template (by heating at 95 °C for 5 min, and
slowly cooling), and 2 nM DNA polymerase in a 10-µl
volume. Reactions proceeded at 37 °C for 3-15 min. Reactions were
stopped by addition of 10 µl of 90% aqueous formamide containing
EDTA (25 mM) and gel sequencing dye solution, followed by
heating in a boiling water bath.
Kinetic Analysis of Incorporation--
Reaction products were
subjected to electrophoresis on 20% polyacrylamide-7 M
urea gels run at 60 watts. After drying the gels, the extent of
incorporation was quantitated with a Fujifilm Fluorescent Image
Analyzer and Image Gauge V3.12 software. Steady-state kinetic
experiments followed the procedures of Creighton et al. (31)
whereby maximum incorporation is kept below 20%. Rectangular hyperbolic fits yielding apparent Vmax and
Km values were calculated using SigmaPlot 2000. Relative values of Vmax/Km for incorrect versus correct nucleotide incorporation were
used to calculate the misincorporation efficiency
(finc).
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RESULTS |
Misincorporation on the Non-adducted Sequence Context III(G) DNA
Template--
Previous studies of pol have used templates that
ranged from 30 to 75 bases in length (17, 18, 28, 32). We first examined our shorter context III(G) 16-mer template to determine its
efficacy as a substrate. In standing start assays with a 12-mer primer,
non-adducted 16-mer template of context III(G) DNA (12/16 DNA) and dCTP
at 1 mM concentration, the primer was mostly extended by
one nucleotide (Fig. 2A). When
all four dNTPs were present at 1 mM further extension
occurred, with limited procession to the end of the template (Fig.
2A). Misincorporation efficiencies (finc) were measured in standing start,
steady-state kinetic assays. Table I
shows similar misincorporation efficiencies of around 2 × 10
3 for all three incorrect nucleotides. These
misincorporation efficiencies are somewhat lower than those reported by
Johnson et al. (17) for the three nucleotides and Matsuda
et al. (32) for G. Matching finc
values are not expected because of differences in assay conditions and
templates. However, observation of misincorporation frequencies that
fall within the range of 102 to 103
previously observed for pol increases our confidence that correct finc values are likely to be obtained from
kinetic studies with 12/16 oligomers containing adducts at the fourth
nucleotide (dG) from the 5'-end of the 16-mer template.
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Fig. 2.
Patterns of dNTP incorporation across from
BaP DE-2 dG adducts in context III(G). For the template sequence
and adduct structures see Fig. 1. A, gel showing
incorporation opposite the fourth nucleotide (G) from the
5'-end of the template strand in the absence of adduct. 0,
control with no dNTP; 4, incorporation in the presence of
all four dNTPs (1 mM each); the other lanes show
incorporation with the individual dNTPs (1 mM) as
indicated. B, gels showing incorporation opposite BaP DE-2
dG adducts in the same position as in A. Labeling of the
lanes is as in A; each dNTP is present at 0.2 mM.
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Patterns of Incorporation in Sequence Context III(G) with BaP DE-2
dG Adducts--
Fig. 2B shows dNTP incorporation across
from each of the four stereoisomeric BaP DE-2 dG adducts, compared with
the non-adducted template. Assays were performed with 0.2 mM dNTP concentrations. It is clear that when any one of
the stereoisomeric BaP DE-2 adducts is present, purine nucleotides are
incorporated far more than either T or the correct C. Misincorporation
of A is most extensive with the trans S adduct.
For each dNTP, more incorporation is seen with the trans-ring opened
adducts compared with the cis adducts with the same R or
S configuration at C-10. Less purine misincorporation occurs at cis adducts than trans adducts, particularly the cis S
adduct, but incorporation of the correct C is also lower. Incorporation at cis R adducts formed from the carcinogenic (+)-BaP DE-2
is greater than incorporation at cis S adducts formed from
the non-carcinogenic ()-BaP DE-2.
The results shown in Fig. 2B for the BaP DE-2 trans
S adduct agree with those obtained by Zhang et
al. (28), who examined only this adduct, in that A is incorporated
the best, but differ significantly in that we observed much higher G
incorporation and much lower T incorporation. Differences between the
assays, such as DNA sequence context or assay buffer composition and
pH, could underlie the differences.
Kinetics of Misincorporation at BaP DE-2 dG Adducts in Sequence
Context III(G)--
Table II shows
Vmax/Km data for
incorporation of individual nucleotides by pol opposite BaP DE-2 dG
adducts in sequence context III(G). In the presence of these adducts,
the highest Vmax/Km values
observed are about two orders of magnitude lower than the
Vmax/Km for incorporation of
the correct C at non-adducted G (Table I). The adducts constitute a
fairly strong block to the enzymatic activity of pol. Notably,
however, the adducts have a blocking effect only on incorporation of a correct C, whereas incorrect base incorporation is either changed very
little or actually enhanced in the presence of adducts. Thus, the
presence of an adduct lowers
Vmax/Km for C incorporation by factors of 1600 to more than 18,000 whereas
Vmax/Km for T incorporation
decreases by 2.2- to 16-fold, G incorporation rises up to 3-fold and A
incorporation rises 1.5- to 7.4-fold.
Typical kinetic data for the trans S dG adduct are shown in
Fig. 3. For all four adducts,
incorporation of the correct C is the lowest of the four dNTPs. In the
assays, there is evidence for inhibition by dNTP concentrations greater
than 2 mM, which limits the amount of dCTP that can be used
to increase incorporation. This makes it difficult to determine a
precise Vmax/Km value for C
incorporation at cis S adducts and precise
finc values for G, A, and T misincorporation.
Still, it is clear that incorporation of purines far exceeds that of
pyrimidines opposite all four adducts in sequence context III(G). The
preference for G misincorporation over correct incorporation of C
ranges from 5.5-fold to more than 50-fold, whereas the preference for A
over C ranges from 20-fold to more than 50-fold.
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Fig. 3.
Kinetics of dNTP incorporation across from
10S trans-ring opened BaP DE-2 dG adducts in sequence
context III(G). A, gels showing incorporation with 0, 20, 50, 100, 200, 500, and 1000 µM dGTP (lanes
1-7); 0, 5, 10, 20, 50, 100, and 200 µM dATP
(lanes 8-14); 0, 50, 100, 200, 500, 1000, and 2000 µM dTTP (lanes 15-21) and dCTP (lanes
22-28). B, hyperbolic Michaelis-Menten plots of the
rates of incorporation using the data obtained from A; units
of Vmax/Km are as in Table
II.
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With all four dNTPs, Vmax/Km
values for purine and T misincorporation are generally greater for
trans ring-opened adducts than cis ring-opened adducts, whether
comparing the pairs trans R versus cis
R or trans S versus cis S
(members of each pair derived from opposite enantiomers of the diol
epoxide) or the pairs trans R versus cis
S or trans S versus cis R
(members of each pair derived from the same diol epoxide enantiomer so that the configuration differs only at C-10).
Vmax/Km values for A, T, and
C incorporation are greatest at trans S adducts, whereas
Vmax/Km for G incorporation
is greatest at trans R adducts. The overall rate of
incorporation (sum of Vmax/Km values) follows the pattern trans R 
trans
S cis R 
cis S. Values of
finc for A and G, however, are much greater for
cis adducts than trans adducts, because of far lower incorporation of C
at cis adducts. Values of finc for G
versus A are similar with all of the adducts except trans
S, whose finc for A is four times
larger than finc for G.
Kinetic Comparisons of Incorporation Across from BaP DE-2 dG
Adducts in Sequence Contexts III(G) and IV(G)--
Preferential
incorporation of purine nucleotides over pyrimidine nucleotides is seen
with both sequence contexts III(G) (5'~CG*A~) (Table II) and IV(G)
(5'~GG*T~) (Table III). But, except
for the trans S isomer, the
Vmax/Km for misincorporation
is far lower and Vmax/Km for
C is unchanged or higher, with the result that
finc values are far lower in context IV(G). This is particularly so with the cis isomers. With the trans R,
cis R and cis S isomers, the decreased preference
for purine misincorporation is due to the decline in
Vmax/Km values in context
IV(G) compared with context III(G) being greater for the purines than the pyrimidines. With the change in sequence context, the
Vmax/Km values for purine
misincorporation drop an order of magnitude for trans R and
cis R adducts and 3- to 5-fold for cis S adducts. With trans S adducts,
Vmax/Km values are greater in
sequence context IV(G), substantially so with G and T incorporation. At the same time, when the sequence is changed to IV(G), C incorporation efficiency declines only 4-fold for trans R adducts and
either stays about the same or rises for the other three adducts.
Consequently, A incorporation at trans S adducts has the
highest efficiency of misincorporation (finc) of
all.
Patterns emerge from trans versus cis comparisons of
Vmax/Km values for
incorporation in sequence context IV(G) that are like the ones seen in
sequence context III(G).
Vmax/Km values for purine
misincorporation are appreciably larger for trans adducts. However,
because C incorporation is so different at context IV(G) adducts,
finc values for G and A for trans adducts are no
longer much smaller than for cis adducts, as was seen in context
III(G). Patterns based on R versus S
comparisons in sequence context IV(G) are not evident.
The effect of changing the sequence from III(G) to IV(G) on T
misincorporation parallels the effect seen with the purines. Vmax/Km for T
misincorporation is higher at trans S adducts, and lower at
the other three. Combined with the aforementioned changes in C
incorporation, the result is a decrease in finc
for T at cis adducts, little change at trans R adducts and a
rise at trans S adducts.
Kinetics of Extension of Primers Containing Correct and Incorrect
Bases Opposite BaP DE-2 dG Adducts in Sequence Context
III(G)--
Table IV summarizes the
Vmax/Km data for extension of
13-mer primers containing either a correct C or a mispaired base
opposite the adducts by incorporation of a correct G opposite the C
immediately 5' to the adduct position in template sequence III(G). Data
for the control, unadducted template are included for comparison.
Notably, extension beyond the adducts is very inefficient, especially
for both the trans adducts as well as the cis S adduct. In
the absence of an adduct, extension of the correctly paired
C-containing primer is favored by a factor of 30 to 100. Strikingly,
however, for all but the cis R adduct, extension of
mispaired primers containing a purine opposite the adduct is favored
( 3-fold) relative to extension of primers containing the correctly
paired C. Thus, once an incorrect base is inserted opposite these
adducts, pol exhibits a bias in favor of extending the mispaired
primer. The cis R adduct constitutes an exception, in that
extension of the correctly paired primer is substantially preferred
over that of primers containing a mispair (although only 2-fold over
extension of a misincorporated A), and this correct extension occurs
with the highest efficiency of any of the adducts.
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Table IV
Extension by pol of (mis)pairs containing BaP DE-2 adducts in
sequence context III(G)
For template sequence see Fig. 1C.
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DISCUSSION |
A Mechanism for Preferential Purine Nucleotide Misincorporation
Across from BaP DE-2 dG Adducts--
All four BaP DE-2 dG adducts
represent significant blocks to C incorporation. In sequence context
III(G), incorporation of the correct C drops as much as 20,000-fold
relative to the corresponding rate in the absence of adduct. In
contrast, the rates of incorporation of the purine nucleotides rise
slightly (less than 10-fold compared with their rates in the absence of
adduct). Consequently, the efficiency
(finc) of A misincorporation at BaP DE-2 dG
adducts in sequence context III(G) (Table II), relative to A
misincorporation in normal DNA (Table I), jumps over four orders
of magnitude from 1.8 × 103 to a range of 20 to over 50 times C incorporation (Table II), and the efficiency of G
misincorporation jumps nearly as much.
We propose the following mechanism for preferential purine
misincorporation opposite the BaP DE-2 dG adducts: 1) Stacking of the
hydrocarbon with the preceding base pair causes the adducted dG to
assume the unusual syn rather than the usual anti torsion angle, with
the stacked hydrocarbon and the syn dG conformation being accommodated
by the open pol active site; 2) the enzyme misincorporates purine
nucleotides opposite this syn dG adduct by Topal and Fresco (33)
purine-purine base pairing, and 3) after purine misincorporation the
adduct remains intercalated in the DNA. Evidence supporting this
mechanism comes from the structural information that follows.
pol exhibits unusually high misincorporation frequencies of
102 to 103 in normal, non-adducted DNA (17,
32). This indicates an active site whose discrimination in favor of
Watson-Crick and other base pairs with similar geometry is considerably
relaxed compared with many other non-proofreading polymerases but is
still 100- to 1000-fold. Crystal structures for members of the Y family
of DNA polymerases (34-37), including the catalytic core of pol
from yeast (35), have been reported. Although Y family polymerases have
a classic DNA polymerase palm structure, the finger and thumb
structures are smaller than usual. An additional structure called a
polymerase-associated domain (35) or little finger (36) is
present. Despite the latter, the Y-family DNA polymerase active site is
more open where the incoming deoxyribonucleotide binds, and the degree
of interaction with the primer-template complex is less than what is
seen with high fidelity DNA polymerases. These features, which are
inferred by homology modeling with T7 DNA polymerase in two studies
(34, 35) and are observed directly in a third (36), could underlie the
relative lack of fidelity exhibited toward non-adducted DNA, and the
ability to accommodate bulky adducts attached to syn-rotated dG and
incorporate a nucleotide across from such lesions (34-37).
The solution NMR structure of a BaP DE-2 trans S dG adduct
bound to the fourth base from the 5'-end of a 13-mer template
(5'-AACG*C~) adjacent to the terminus of a 9-mer primer strand
provides a model for a partially extended DNA duplex as "seen" by a
polymerase. This structure shows that prior to insertion of the
complementary nucleotide, the adducted dG is rotated anti to syn to
allow the hydrocarbon moiety to stack with the base pair formed by the
3'-terminal primer base and the base immediately 3' to the modified dG
on the template strand (38). After a complementary C is incorporated opposite the BaP DE-2 trans S dG adduct, the adducted dG
assumes the normal anti conformation and the adduct swings out into the minor groove (39). This same groove-bound, anti conformation of the
adduct is maintained upon further extension of the primer chain beyond
the adduct site, as is shown by the structure of a fully complementary
11-mer duplex (40) with the same local ~CG*C~ sequence around the adduct.
There are no NMR data for mismatched duplex structures
corresponding to incorrect purine nucleotide insertion
opposite BaP dG adducts; however, substantial UV red shifts are
observed for the long-wavelength pyrene band of duplexes containing
purine mismatches opposite BaP trans R and trans
S dG adducts in the same ~CG*C~ context (41), from 346 nm (when C is paired with the adducted G) to around 350 nm in the G and
A mispaired duplexes. These shifts are indicative of base stacking, and
suggest that the hydrocarbon remains intercalated (41), with the
adducted dG presumably still in the syn conformation, after an
incorrect purine nucleotide is inserted opposite the adduct
and the primer is further extended.
If the adducted dG is in the syn conformation before and after
polymerization then presumably it is also syn when the incoming nucleotide binds and is incorporated. A syn glycosidic torsion angle of
the adducted dG in the template allows purine-purine base pairing of
the type proposed by Topal and Fresco (33). Molecular modeling (42) of
the BaP DE-2 trans S adduct in a 13-mer template, 10-mer
primer structure (the 13-mer template and 9-mer primer described above
with the primer extended to include A across from the adduct) suggests
that the size and shape of the (syn-adducted-G)·(anti-A) pair very
much resembles that of a normal T·A pair. The 100- to 1000-fold
preference for base pairs having the approximate size and shape of a
Watson-Crick base pair could also cause the enzyme to select
syn-adducted G paired with anti-A over pairing with the correct C. The
observed red shifts (41) increase (more favorable base stacking) in the
same order as the present
Vmax/Km values for pol
misincorporation of purines at trans adducts.
Even though an NMR structure could not be determined (38) when the
trans S dG adduct above is replaced by a trans R
dG adduct, it is likely that the pol preference for forming
(adducted-G)·(purine) mispairs with the BaP trans R adduct
can be explained by a mechanism similar to that described above for the
trans S adduct. UV spectra of oligonucleotide duplexes
containing the trans R dG adduct mispaired with purines show
significant red shifts of the long wavelength absorbance (41),
consistent with base stacking and syn glycosidic rotation, and are
indicative of a structure analogous to that observed with the trans
S dG adduct.
Structural analysis of either a cis R or cis S
BaP DE-2 dG adduct placed in the same position as the trans adducts
just described, either before or after a correct or incorrect
incorporation of nucleotide has yet to be reported. In fully duplex
DNA, the guanine base bearing either a cis R (43) or a cis
S (44) BaP adduct is displaced out of the DNA helix as is
its complementary C, and neither NMR nor UV studies with purine-purine
mispairs of cis adducts are available. Thus no structural information
exists that might explain preferential purine incorporation
versus C incorporation at the present cis adducts; however,
as in the case of the trans adducts, the observed preference for purine
misincorporation presumably could also result from anti to syn rotation
of the adducted dG and Topal and Fresco (33) base pairing.
8-Oxo-dG forms stable G·A pairs in DNA with the G rotated anti to syn
(45), resulting in G to T transversions (A misincorporation) with
pol
(46). In mammalian cells, G T transversions also commonly
occur at N-(deoxyguanosin-8-yl)-acetylaminofluorene
adducts depending on the DNA sequence (47) and are attributed to the adducted dG adopting a syn conformation (48). pol, however, mostly
incorporates the correct C across from these two types of C-8 G adducts
(16, 49), suggestive that with these types of adducts the active site
of pol forces the G to rotate back to the anti conformation. It is
interesting that this occurs with these C-8 dG adducts but not with the
present BaP DE-2 N2-dG adducts.
Effect of Sequence Context on Misincorporation--
The most
pronounced sequence effect seen in this study is the sharply lower set
of Vmax/Km values for
misincorporation observed with sequence context IV(G) (Table III)
compared with sequence context III(G) (Table II), seen with all adducts
except for trans S BaP DE-2. For the other adducts in
sequence context IV(G), the decrease in the rate of purine
misincorporation is more than an order of magnitude with the cis
R adduct, about an order of magnitude with the trans
R adduct and 3- to 5-fold with the cis S adduct.
The Vmax/Km values for C
incorporation decrease by small amounts with two of the adduct isomers
and rise with the other two. The higher rate of C incorporation in
sequence context IV(G) compared with context III(G) could arise from
misalignment using the next 5'-template G in place of the adducted G.
With sequence context IV(G) the
Vmax/Km for misincorporation
of A at the trans S adduct is the largest for any
nucleotide-adduct combination and is 18-fold greater than for A
misincorporation at the trans R adduct. This observation is
of special interest in light of the fact that the trans S is
the predominant adduct formed on DNA (50) by the highly carcinogenic
(+)-BaP DE-2 isomer whereas trans R is the predominant
adduct formed by the weakly or non-carcinogenic ()-BaP DE-2 isomer
(26).
A significant finding of this study is that the sequence context effect
on the efficiency of base misincorporation depends on the specific
adduct. The present two sequences differ in both the 3'- and 5'-nearest
neighbors to the adducted G. The 3'-nearest neighbor is A in sequence
context III(G) and T in sequence context IV(G). Specific stacking
interaction between an adduct and the preceding (3') base pair is
suggested by NMR analysis (38) of a partial template·primer duplex in
which the 3'-nearest neighbor to the adducted G is C (see above). Small
differences in stacking geometry between the adduct and different
3'-nearest neighbor bases and their complements could account for the
observed differences in misincorporation frequency between templates
III(G) and IV(G). Specific effects of the 5'-nearest neighbor are also
possible, such as template slippage leading to enhanced correct
insertion of C with template IV(G) as mentioned above or changes in
stacking depending on the identity of the 5'-base. Our observed
dependence of misincorporation on sequence suggests that it will be
fruitful to focus bypass studies of misincorporation on specific
proto-oncogene and tumor suppressor sequences.
Mutational Spectra of BaP DE-2 dG Adducts in Mammalian DNA--
It
is of considerable interest to establish what role pol plays in
determining the mutational spectrum that has been observed for PAHs in
mammalian cells. The preponderant products of in vitro reaction of BaP diol epoxides with dG in DNA are trans ring-opened adducts formed at the exocyclic nitrogen of dG. Upon reaction of
()-BaP DE-2 with calf thymus DNA in vitro, trans
R and cis S dG adducts are formed in the ratio of
~6:1 (50). At both types of adduct, pol strongly favored
misincorporation of purines, with both G and A being misincorporated
about equally and 5 to 20 times the rate for T misincorporation. Once
purine misincorporation occurred, such mispairs were extended somewhat
better than the correctly paired primers for three out of the four
adducts studied in sequence III(G). However, the extension of these
mispairs was still quite inefficient
(Vmax/Km generally about 10- to 20-fold smaller than
Vmax/Km for purine
incorporation; Tables II and IV), suggesting that if purine
misincorporation by pol plays a role in mutagenesis induced by BaP
DE adducts, an additional polymerase or polymerases are likely to be
required for full extension of the damaged DNA to give an observed
mutagenic event. Precedent for such a mechanism is provided by the
observation that pol
and pol
act sequentially to bypass abasic
sites as well as the (6-4) T-T photoproduct (51).
The relative lack of T misincorporation by pol contrasts with
limited data (52) on the mutational spectrum of ()-BaP DE-2 in the
HPRT gene in Chinese hamster V-79 cells, in which G
C, G T, and G A mutations (misincorporation of G, A, and T,
respectively) occurred to similar extents (1.5:1.0:0.8). Far more
information is available about the spectrum of mutations at G produced
by (+)-BaP DE-2 in mammalian cells. In the HPRT gene in V-79
cells, the relative proportions of the three possible base
substitutions at G are virtually independent of dose (53). At three
different doses of (+)-BaP DE-2, G T transversions accounted for
67-75%, G C transversions for 18-21%, and G A transitions
for 7-11% of the total mutations (53). The use of repair-deficient
Chinese hamster V-H1 cells (54) resulted in little change in the
relative mutational frequencies at two different doses: 60-63% of G
mutations were G T transversions, 20-23% were G C
transversions, and 15-19% were G A transitions. These frequencies
of misincorporation are consistent with our pol results described below.
The spectrum of mutations produced in the ras proto-oncogene
was determined (55) in mice whose skins were painted with the parent
hydrocarbon, BaP. When forming bay-region diol epoxides from the (+)-
and ()-enantiomers of BaP 7,8-dihydrodiol, liver microsomes from
3-methylcholanthrene-treated rats produce predominantly (+)-BaP DE-2
and much less of the other three diol epoxide isomers (56). Despite the
different cell types and the overlay of BaP metabolism in the skin
experiments, the mutational pattern is quite similar for BaP on mouse
skin and (+)-BaP DE-2 in hamster cells in culture. In mouse skin, the
proportion of G T transversions is slightly higher (83%), and G
C transversions (13%) and G A transitions (4%) are
correspondingly lower than what was seen in the cultured cell studies.
Misincorporation by pol at (+)-BaP DE-2 adducts fits the above
mutational spectra well. In vitro reaction of (+)-BaP DE-2 with calf thymus DNA gives trans S and cis R
adducts in the ratio of ~40:1 (50). Therefore, it is reasonable to
assume that mutations arising at G in mammalian cells are likely to
arise largely from the trans S adducts formed in multiple
sites in the DNA, and the effect of cis R adducts may be
ignored. Misincorporation of A, G, and T by pol at trans
S adducts would give rise to G T, G C, and G A
substitutions, respectively. The proportions of A, G, and T
misincorporation in sequence contexts III(G) and IV(G) (calculated from
the data shown in Tables II and III, respectively) are A, 76% and
58%; G, 19% and 31%; and T, 5% and 10%. When extension of the
appropriate mispairs is also taken into account by comparison of the
products
(Vmax/Km)inc·(Vmax/Km)ext,
the proportions of A, G, and T mispairs that are formed and also
extended by one base beyond the lesion in context III(G) are 65%,
32%, and 3%, respectively. These relative misincorporation and bypass efficiencies coincide well with the mammalian cell and mouse skin mutational spectra. Although another Y-family DNA polymerase pol
can
also bypass BaP DE dG adducts (57), it does so in a largely error-free manner and is thus unlikely to be responsible for
mutagenesis induced by these lesions (58). It is tempting to speculate
that purine misincorporation opposite BaP DE adducts by
error-prone pol may play a role in generating the pattern
of mutations seen in mammalian cells containing these DNA lesions.
Opposite Benzo[a]pyrene 7,8-Diol
9,10-Epoxide Deoxyguanosine Adducts*
,
,
TOP
ABSTRACT
INTRODUCTION
Analytical Methods and Pharmacology
(Wainer, I. W.
, and Drayer, D. E., eds)
, pp. 271-296, Marcel Dekker, Inc., New York, NY
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