From the Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-4660
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ABSTRACT |
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Base excision repair of dimethyl sulfate induced
N-methylpurines (NMPs) was measured in a yeast
minichromosome that has a galactose-inducible GAL1:URA3
fusion gene, a constitutively expressed HIS3 gene, and
varied regions of chromatin structure. Removal rates of NMPs varied
dramatically (>20-fold) at different sites along three selected
fragments encompassing a total length of 1775 base pairs. Repair of
NMPs was not coupled to transcription, because the transcribed strands
of HIS3 and induced GAL1:URA3 were not repaired
faster than the nontranscribed strands. However, the repair rate of
NMPs was significantly affected by the nearest neighbor nucleotides.
Slow repair occurred at NMPs between purines, especially guanines,
whereas fast repair occurred at NMPs between pyrimidines. NMPs between
a purine and pyrimidine were repaired at moderate rates. Moreover, a
rough correlation between nucleosome positions and repair rates exists
in some but not all regions that were analyzed.
Subtle alterations in DNA molecules, such as oxidized or
alkylated bases, are repaired by the base excision repair
(BER)1 pathway (reviewed in
Refs. 1 and 2). BER is initiated by specific DNA glycosylases that
release the damaged base. There are two families of alkylated base DNA
glycosylases (3). Although their primary sequences and
three-dimensional structures are unrelated, they may have common themes
in recognition of alkylated bases and catalysis of glycosylic bond
cleavage (3). Crystal structures of the human AAG protein (3) and the
Escherichia coli AlkA protein (4, 5) suggest that the
glycosylases flip the target base out of the DNA duplex into the active
site cleft of the enzymes. The "flipping out" may occur in a later
step during recognition and/or incision of damaged bases (6).
Alkylation damage to DNA occurs frequently in nature and can be
introduced either by alkylating agents present in the environment or by
erroneous alkylation induced by the natural methyl donor S-adenosyl-methione (7). Simple methylating agents such as dimethyl sulfate (DMS) produce a variety of damaged bases in DNA of
which N-methylpurines
(N7-methylguanine and
N3-methyladenine) or NMPs constitute
approximately 90% of the alterations (8). In Saccharomyces
cerevisiae, the repair of NMPs occurs mainly through the BER
pathway, although nucleotide excision repair (NER) may play a role if
the BER pathway is abolished (9). Initiation of this pathway in
S. cerevisiae is by the action of the small (34 kDa) protein
MAG DNA glycosylase (10).
Although the repair of thymine glycols is coupled to transcription in
both yeast and mammalian cells (11, 12), a link between transcription
and BER of other lesions has not been well established. It was found
that the alkali-labile sites (representing NMPs) induced by
methylnitrosourea are preferentially repaired within the active insulin
gene of rat insulinoma cells (13). However, no preferential repair of
these lesions induced by DMS was found in the actively transcribed
DHFR gene of Chinese hamster ovary cells (14-16) or in the
transcribed strand of the PGK1 gene in human cells (17).
These studies raise several questions about BER in intact cells. As a
damaged base is flipped out by a DNA glycosylase during BER, do
neighboring nucleotides affect the rate of BER? Is there a coupling
between transcription and BER of NMPs in yeast? Futhermore, as
modulation of NER by chromatin structure has been well established (18), does nucleosome positioning also modulate BER? To address these
questions, we studied BER of NMPs in a minichromosome in intact yeast cells.
Plasmid and Yeast Strains--
Strain RGY1 was made by
transforming strain Y452 (MAT DMS Treatment and DNA Repair Incubation--
RGY1 cells were
grown at 30 °C in minimal medium containing 2% glucose or 2%
galactose to late log phase (A600 ~ 1.0). The cultures were mixed with DMS to give a final concentration of 0.05%
(v/v). After 5 min of incubation at room temperature, the cells were
washed twice with either ice-cold 2% glucose or 2% galactose and
resuspended in the same solutions containing 100 mM
hydroxyurea to prevent DNA replication during repair incubation (20).
One-tenth volume of a solution containing 10% yeast extract and 20%
peptone was added to the DMS-treated cultures. After 0, 1, 2, and
4 h of repair incubation at 30 °C, an aliquot was removed, and
plasmid DNA was isolated as described previously (21).
Analysis of NMP Repair at the Nucleotide Level in
YRpSO1--
A biotinylated oligonucleotide and streptavidin magnetic
bead-facilitated end-labeling technique was used (22, 23). Briefly, isolated plasmid DNA was cut with restriction enzyme(s) to release the
three fragments that were analyzed. DdeI and StyI
were used to release the 678-bp GAL1-URA3 fragment,
StuI was used to release the 717-bp URA3-3'
fragment, and BstNI and BstXI were used to release the 659-bp HIS3 fragment (see Fig. 1). The
restricted DNA was cleaved at NMP sites by incubation in 1 M piperidine at 90 °C for 30 min (24). One strand of the
cleaved fragments was annealed to biotinylated oligonucleotides
(sequences available upon request). The annealed fragments were
attached to streptavidin magnetic beads (Dynal, Inc) and labeled using
[ General Features of YRpSO1--
YRpSO1 is a high copy number
(~50 copies/cell), autonomously replicating yeast plasmid 4263 bp in
length (Fig. 1). It contains (a) the ARS1 orgin of replication, flanked by the
3' end of the TRP1 gene (Fig. 1, open arrowhead)
and the upstream region of the GAL3 gene (Fig. 1, near
nucleosome 9), (b) a galactose-inducible GAL1:URA3 fusion gene, (c) a constitutively
expressed HIS3 gene, and (d) the 5' ends of the
PET56 and DED1 genes of yeast (19). Moreover, a
sequence of unknown function (UNF) lies downstream of the
DED1 fragment. Twenty-two nucleosomes (Fig. 1, small
numbered circles) are distributed in four regions, interrupted by
four nuclease-sensitive gaps
(19).2 Following galactose
induction, four nucleosomes (Fig. 1, nucleosomes 19-22) in
the UNF-GAL10 region are destabilized or rearranged. Upon
galactose induction, the level of GAL1:URA3 RNA increases dramatically (>150-fold) from essentially zero in glucose medium (25).
However, except for the regions mentioned above, no change in
nucleosome structure is observed in the minichromosome (including the
GAL1:URA3 fusion gene) in galactose cultures (19),
indicating that only a few of the fusion genes are transcriptionally
active at any one time.3 This
fact presumably reflects insufficient GAL4p for binding to the
UASg of the GAL1-10 promoter contained in the
multicopy plasmid.
Induction and Repair of NMPs at Individual Sites in
YRpSO1--
Three fragments of YRpSO1 (GAL1-URA3
(DdeI-StyI), URA3-3'
(StuI), and HIS3
(BstNI-BstXI)) were chosen for analysis based on their proximity to the GAL1:URA3 and HIS3 genes
(Fig. 1). Representative gels showing the incidence and repair of NMPs
are shown in Figs. 2 and
3. As can be seen, the induction of NMPs
was primarily at G residues (i.e. compare 0 h and
G lanes). This is in agreement with past reports (reviewed
in Ref. 8), as N7-methylguanines induced by DMS
account for >80% of the total adducts in DNA. We note that galactose
caused no obvious change in the induction of NMPs in the
UASg and the promoter regions of the GAL1:URA3
fusion gene (Fig. 3). In contrast, changes in induction levels are
observed in these regions of the single copy genomic GAL1
gene.4 As mentioned above, this may reflect
insufficient amounts of GAL4p bound to the UASg during
galactose induction.
The band intensities at 460 total sites in the three fragments,
following 1, 2, and 4 h of repair, were quantified using peak deconvolution. A decrease in band intensities indicates that NMPs at
respective sites are removed. This removal reflects the complete process of BER rather than just the excision of NMPs, as the apurinic sites generated from the excision of NMPs are also alkali-labile and
unligated BER intermediates would (by themselves) be DNA single strand
breaks. We detected no obvious DNA single strand breaks at any time
during repair incubation if the samples were not treated with alkaline
piperidine, presumably because the post-excision repair process is very
fast. An example of the percentage of NMPs remaining in each strand
after 2 h of repair incubation is
shown in Fig. 4. As implied by this data,
the time course of repair was similar at most sites in the two cultures
(Fig. 4).
Effect of Transcription on NMP Repair--
Removal of NMPs does
not appear to be coupled to transcription, because the transcribed
strands of the constitutively expressed HIS3 gene and the
galactose-induced GAL1:URA3 fusion gene are not repaired
significantly faster (Figs. 2-4). The fraction of NMPs remaining at
each site in glucose and galactose cultures were directly compared to
examine more subtle changes when transcription is induced (Fig.
5). These results indicate that a few
sites in the GAL1 promoter (nucleotides 462, 502, and 507;
also see bands marked with bars in Fig. 3) and
downstream of the GAL1:URA3 fusion gene (nucleotides 1684, 1720, and 1741) are repaired faster in galactose cultures. Because
these sites are outside of the transcribed regions, this result may
reflect subtle disruption of nucleosomes in these two regions that was
undetected by nucleosome mapping (19).
Effect of DNA Sequence on NMP Repair--
In both glucose and
galactose cultures, the repair rate of NMPs varied dramatically along
the three fragments (Figs. 2-4). Analysis of these variations with DNA
sequence yielded an unexpected correlation between NMP sequence
location and removal by BER. Most NMP sites located between pyrimidines
(e.g. bands marked by small arrows in
Figs. 2 and 3) are repaired much faster than NMPs between purines, although exceptions to this trend do exist (e.g.
bands marked by asterisks in Figs. 2 and 3).
To determine how general this sequence effect may be, we compared
the repair rates of NMPs at 371 different sites that were well
separated (or easily deconvoluted) bands on sequencing gels. As can be
seen in Table I, in both glucose and
galactose cultures, NMPs between two purines (especially two Gs) are
repaired most slowly, NMPs between two pyrimidines (C or T) are
repaired most rapidly, and NMPs between a purine and pyrimidine are
repaired at a moderate rate. In contrast, second adjacent nucleotides
(or a combination of the first and second adjacent nucleotides) have little (or no) effect on repair of NMPs (data not shown).
Effect of Nucleosome Positioning on NMP Repair--
As can be
deduced from Fig. 4, a rough correlation between nucleosome positions
and repair rates exists for both strands of the GAL1:URA3
fusion gene in the nucleosome regions approximately centered at
nucleotides 1300 and 1460. In these regions, most sites within
nucleosomes were repaired slower, whereas most sites in the
internucleosome regions were repaired faster. A rough correlation can
also be deduced for the TS of the GAL1:URA3 gene in the
nucleosome region approximately centered at nucleotides 430 and 610 (Fig. 4). Because nearest neighbor nucleotides can dramatically affect the repair rate of NMPs, the percentages of NMPs remaining at the 371 NMP sites after each repair time were also normalized by the mean
values of the corresponding sequence contexts (Table I). After this
normalization, the correlation between repair rates of NMPs and
nucleosome positioning was somewhat more evident (data not shown).
However, no obvious modulation of nucleosome positioning on repair was
seen in the HIS3 gene and in the 5' region of the NTS of the
GAL1:URA3 fusion gene (Fig. 4).
We observed no obvious preferential removal of NMPs in the
transcribed strands of the galactose-induced GAL1:URA3
fusion gene and in the constitutively expressed HIS3 gene of
YRpSO1 (Figs. 2-5). Conversely, we have found a coupling between NER
of UV-induced cyclobutane pyrimidine dimers and transcription in both
of these genes.3 These results agree with those found for
BER of NMPs in the DHFR gene of Chinese hamster ovary cells
(14-16) and for the PGK1 gene in human cells (17) but
differ from those obtained for the insulin gene in rat insulinoma cells
(13). At present, the reason for this discrepancy is unclear, but it
may reflect a difference in the mode of gene expression between these
cells and/or gene types.
In contrast to the lack of correlation with transcription, repair of
NMPs is dramatically influenced by nearest neighbor nucleotides (Table
I). These nucleotides could have a significant effect on the energy
required for flipping out NMPs during recognition and incision by DNA
glycosylases (3-6). Indeed, theoretical calculations show that the
stability of base stacking follows the order: purine-purine (G-G
slightly > A-A) We observed a rather weak correlation between NMP repair and nucleosome
positioning (Fig. 4).4
However, this does not necessarily mean that histone binding to DNA has
little effect on NMP repair. Although this effect has not been well
studied, many reports indicate that NER is modulated by nucleosomes
(reviewed in Ref. 18). Therefore, this weak correlation between NMP
repair and nucleosome positioning in YRpSO1 may suggest that the effect
of nucleosome positioning is partially "masked" by effects of other
factors on repair of NMPs.
Finally, recently the more sensitive ligation-mediated polymerase chain
reaction method was used to study BER of NMPs in human cells (17).
These authors also found extreme variability in BER rate at different
positions and no transcription-coupled repair in the PGK1
gene. Thus, the observations in the present work appear to be testable
in mammalian cells. Clearly, it will be of interest to determine
whether repair of NMPs in mammalian cells is modulated by the same
pattern of nearest neighbor nucleotides as we found in yeast.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52 his3-1 leu2-3 leu2-112
cir°) with plasmid YRpSO1 (see Fig. 1). This plasmid was
constructed and characterized by Drs. Stephano Omari and Fritz Thoma at
the Swiss Federal Research Institute (ETH-Hönggerberg) in
Zürich, Switzerland (19).
-32P]dATP (NEN Life Science Products) and SequenaseTM
(Amersham Pharmacia Biotech). The labeled fragments were eluted,
resolved on sequencing gels, and exposed to PhosphorImager screens
(Molecular Dynamics). The band intensities in the gels were quantified
using ImagQuaNT (Molecular Dynamics) and PeakFit 4.0 (SPSS, Inc.)
deconvolution software as described (23). Sequence markers were
generated from polymerase chain reaction fragments, as described
previously (22, 23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic drawing of YRpSO1. DNA
elements are indicated on the large circle. Small
numbered circles denote approximate nucleosome positions.
Shaded circles (numbered with 19-22) denote nucleosomes
that are destabilized or rearranged in galactose cultures. The
hatched circle (number 8) indicates space for a nucleosome,
although the footprint was unclear. The open box in the
GAL1-10 region denotes the UASg. Small
solid ellipse demarcates ARS1 consensus sequence
(A-element). The open arrowhead downstream of
URA3 denotes the 3' end of the TRP1 gene.
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Fig. 2.
Phosphorimages of sequencing gels showing
repair of NMPs at specific sites in the URA3-3'
fragment. Lanes labeled CT, AG,
and G are sequence markers generated through a modified
procedure of Maxam-Gilbert sequencing. Lanes labeled
U are untreated controls. Small arrows and
asterisks (marked with nucleotide positions) on the
right side of each gel denote NMP sites between pyrimidines
that were repaired rapidly and slowly, respectively. The nucleotide
positions are clockwise from the unique EcoRI site of YRpSO1
(see Fig. 1). Schematic diagrams of the minichromosome region for this
fragment are shown on the left side of each gel. The
shaded ellipses represent approximate nucleosome positions,
the dark thick arrows denote the transcribed region of the
URA3 gene, and the shaded lines denote the 3' end
of the TRP1 gene. Left panel, TS for the
URA3 gene. Right panel, NTS for the
URA3 gene.
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Fig. 3.
Phosphorimages of sequencing gels showing
repair in the GAL1-URA3 and HIS3
fragments. Lanes labeled CT,
AG, and G are sequence markers generated through
a modified procedure of Maxam-Gilbert sequencing. Lanes
labeled U are untreated controls. Arrows and
asterisks (marked with nucleotide positions) on the
right side of each gel denote NMP sites between pyrimidines
that were repaired rapidly and slowly, respectively. Short thin
bars (nucleotides 462, 502, and 507) mark the sites that were
repaired more rapidly in galactose cultures. The nucleotide positions
are clockwise from the unique EcoRI site of YRpSO1 (see Fig.
1). Left panel, GAL1-URA3 fragment (TS for the
fusion gene) that encompasses the UASg and part of the
promoter of GAL1. Roman numbers denote the four
GAL4p binding sites in the UASg of GAL1.
Right panel, the HIS3 fragment (TS) that
encompasses the 5' end of the PET56 gene and the promoter
and 5' coding region of the HIS3 gene.
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Fig. 4.
The percentage of NMPs remaining at specific
sites in the three fragments after 2 h of repair incubation.
Open circles and solid triangles represent the
percentages of NMPs remaining at individual sites in glucose and
galactose cultures, respectively. For each fragment, between the two
panels (for TS and NTS, respectively) is shown a schematic diagram of
the minichromosome region. The large shaded ellipses
represent the approximate nucleosome positions. The wavy
arrows denote the major transcription start sites for the
GAL1:URA3 fusion gene and HIS3 gene,
respectively. The small solid ellipses denote the TATA boxes
for the GAL1, PET56, and HIS3 genes.
The open boxes with Roman numerals denote the GAL4p binding
sites in the UASg. The numbering is clockwise
from the unique EcoRI site of YRpSO1 (Fig. 1).
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Fig. 5.
Comparison of NMP repair between glucose and
galactose cultures along the three fragments. Values shown are the
differences in the percentages of NMPs remaining at individual sites in
the TS (solid triangles) and NTS (open circles)
for glucose and galactose cultures following 2 h repair
incubation. Symbols in the schematic diagrams are defined in the legend
of Fig. 4.
Effect of nearest neighbor nucleotides on repair of NMPs
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
purine-pyrimidine > pyrimidine-purine > pyrimidine-pyrimidine, with a 2-kcal/mol
spread between the least stable and most stable base pairs (26). Thus,
it can be predicted that NMPs between purines (especially Gs) require
the most energy to be flipped out, whereas those between pyrimidines
require the least energy. These predictions fit exactly with our
results (Table I). However, it should be noted that some NMPs located
between pyrimidines were also repaired slowly (e.g.
bands marked by asterisks in Figs. 2 and 3). The
slow repair at these sites may reflect other factors in chromatin
(e.g. nonhistone DNA-binding proteins) that may also affect
repair of NMPs.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ranjan Gupta for creating the yeast strain RGY1, Dr. Fritz Thoma for supplying the plasmid YRpSO1 and helpful discussions, and Dr. Louise Prakash for providing the yeast strain Y452. We also thank members of the Smerdon laboratory, particularly Maria Meijer and Joseph Kosmoski, for critical discussions.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grant ES04106 from the National Institute of Environmental Health Sciences.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.
To whom correspondence should be addressed. Tel.: 509-335-6853;
Fax: 509-335-9688; E-mail: smerdon{at}mail.wsu.edu.
2 F. Thoma, unpublished results.
3 S. Li, R. Gupta, M. Meijer, M. Livingstone-Zatchej, F. Thoma, and M. J. Smerdon, submitted for publication.
4 S. Li and M. J. Smerdon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: BER, base excision repair; bp, base pair(s); DMS, dimethyl sulfate; NMP, N-methylpurine; NER, nucleotide excision repair; TS, transcribed strand; NTS, nontranscribed strand.
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