From the
Using radioimmunoassays, we examined rates of removal of
UV-induced pyrimidine-pyrimidone) photoproducts ((6-4)PDs) and
cyclobutane pyrimidine dimers (CPDs) from 146-base pair nucleosome core
DNA (and 166-base pair chromatosome DNA) of confluent human diploid
fibroblasts. Dose-response experiments indicate that the yield of)PDs
in core DNA is about 30% that of CPDs in the UV dose range of
0-200 J/m
There are two major classes of stable photoproducts induced in
DNA by ultraviolet (UV) radiation, namely cis-syn cyclobutane
pyrimidine dimers (CPDs)
The marked difference in repair rate of these two photoproducts in
mammalian cells may reflect (a) their different structures in
DNA(10, 11) , (b) their different distributions
in chromatin(12, 13) , and/or (c) the
involvement of different recognition proteins for these
lesions(14) . Indeed, at the nucleosome level of chromatin, CPD
formation is significantly modulated with a 10.3 base average
periodicity in nucleosome cores(15, 16) , while
(6-4)PDs are much more randomly distributed in these
regions(12) . In addition, whereas a similar yield of CPDs/bp is
found in nucleosome core and linker DNA(17, 18) , the
yield of)PDs/bp is greater in nuclease-``sensitive'' DNA than
nuclease-``resistant'' DNA of chromatin(13) .
Recently, Szymkowski et al.(14) reported that in
cell extracts, using naked DNA as a template and repair synthesis as a
measure of excision repair,)PDs are repaired much more rapidly than
CPDs. Thus, even in vitro (and, presumably, in the absence of
nucleosomes))PDs are repaired much more rapidly than CPDs by the NER
complex. This result suggests that the repair complex can locate and
incise)PDs more efficiently than CPDs in naked DNA. In intact cells,
this could be facilitated by the difference in nucleosome distribution
within chromatin of these two photoproducts (discussed above). Indeed,
rapid repair of)PDs in intact cells could (at least partially) reflect
their preferential location in regions of chromatin that are more
accessible to repair enzymes. Repair of)PDs in nucleosome cores, which
may be less accessible to these enzymes, may occur more slowly. Such
``longer lived lesions'' could yield hot spots for
mutagenesis. Therefore, we studied the rate of removal of both types of
photoproducts at the nucleosome level of chromatin in intact human
cells. Antibodies specific to each photoproduct were used to detect the
induction frequency of CPDs and (6-4)PDs and used to measure
their removal from nucleosome core (and chromatosome) DNA.
Human diploid fibroblasts (strain AG1518) were irradiated at
various doses of UV to determine the relative yield of)PDs and CPDs
within nucleosome core DNA. Nucleosome core (146 bp) and chromatosome
(166 bp) DNA was isolated by preparative gel electrophoresis from
nuclei digested with micrococcal nuclease. As shown
previously(22) , when human cell nuclei are digested in low
ionic strength isotonic buffer, micrococcal nuclease liberates well
defined bands of core and chromatosome DNA (Fig. 1). These
bands were cut out of the gel and electroeluted, yielding only
nucleosome core (or chromatosome) DNA.
Finally, the ratio of
each photoproduct was determined in core DNA and genomic DNA as a means
of determining the relative amounts of each lesion in nucleosome core
DNA and ``non-core DNA'' in irradiated human cells. The
ratios were determined from RIA assays (i.e. 50% inhibition
values) with purified genomic DNA, which was fragmented with PstI, and nucleosome core DNA isolated from the same cells.
Ratios of 0.66 (for CPD) and 0.44 (for (6-4)PD) were obtained (), indicating that the induction of)PDs in core DNA
(relative to genomic DNA) is less than that of CPDs. This result is in
general agreement with Mitchell et al.(13) , although
these authors observed a ratio for CPDs closer to 1.0.
We have shown that the rates of repair of the two major
(stable) UV photoproducts in DNA (CPD and)PD) are markedly different in
nucleosome core (and chromatosome) regions of human chromatin. Even
during the early ``rapid phase'' of CPD removal (reviewed in
Ref. 29; see also Fig. 3),)PDs are removed
These results indicate that nucleosome structure does not
markedly retard repair of)PDs in intact human cells. It is possible
that the repair rate of)PDs is somewhat slower in these regions
(relative to nucleosome-free DNA), but it is clear that the rate is far
more rapid than that of CPDs. Furthermore, the rates of removal of)PDs
and CPDs we observe in confluent human fibroblasts are similar to those
reported for the genome overall in human fibroblasts synchronized in S
phase(32) . One possibility is that (6-4)PDs are
recognized by a protein that does not recognize (or has a lower
affinity for) CPDs that ``tags'' these lesions and initiates
NER. Indeed, the human-damaged DNA-binding protein XPE has been
observed to bind)PD with higher affinity than CPDs in synthetic DNA
substrates(33) . In addition, a) photolyase has been observed in Drosophila(34) . Thus, proteins do exist that
distinguish between these UV photoproducts.
The structure of)PDs in
DNA may render these lesions more accessible to repair enzymes than
CPDs. The adjacent pyrimidine rings in cis-syn cyclobutane
dimers are predicted to be nearly coplaner(11) , whereas the
pyrimidine-pyrimidone rings in)PDs are approximately
perpendicular(10) . This difference in local DNA structure may
significantly enhance incision and removal of)PDs, compared with CPDs.
It should be noted, however, that in this report we have measured
repair of each photoproduct within nucleosome cores, and these
regions presumably would not be isolated by our techniques if)PDs
caused a major disruption of histone binding (with a concomitant
increase in digestion by micrococcal nuclease). Thus, whatever the
difference between)PD and CPD recognition, it occurs at photoproducts
formed within folded DNA of nucleosome cores.
We thank Sylvia Hering for assistance with the cell
culture and UV irradiations. We also thank Drs. Bonnie Baxter and David
Brown for critically evaluating this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. Repair experiments indicate that, at 40
J/m
,)PDs are removed much faster (
75% in 2 h) from
nucleosome core (and chromatosome) DNA than CPDs (10-15% in 2 h).
A slow rate of removal of CPDs is also observed when the UV dose is
reduced to 10 J/m
(i.e. even when the level of
CPDs is less than that of)PDs at 40 J/m
). These results
indicate that (a) the accessibility of repair proteins to)PDs
in nucleosomes is markedly different than their accessibility to CPDs
and/or (b) repair enzymes are much more efficient at incising
and removing)PDs than CPDs in human chromatin.
(
)and
pyrimidine-pyrimidone) dimers ((6-4)PDs)(1) . The most
prominent of these are CPDs, which are classic examples of mutagenic
and carcinogenic lesions in DNA(2, 3) . However, even
though the overall yield of CPDs is about 3-fold greater (on average)
than)PDs in genomic DNA at physiologic doses of UV light, the
contribution of (6-4)PDs to UV-induced mutagenesis can be
significant in certain cell types(3, 4, 5) .
Indeed, the yield of)PDs at specific sites within mammalian
chromatin can be much higher than their yield in free DNA(6) .
This could be one of the factors leading to mutagenic ``hot
spots'' in certain sequences(3, 4) . Furthermore,
although both lesions are removed by nucleotide excision repair (NER),
the overall repair of)PDs in genomic DNA is much more rapid than CPDs
in virtually all cells examined (e.g. see Refs. 7-9).
Cell Culture, Labeling, and UV Irradiation
Human
diploid fibroblasts (strain AG 1518) were purchased from the Human
Genetic Mutant Cell Repository (Camden, NJ) and grown in
Dulbecco's modified Eagle's medium supplemented with 5%
fetal bovine serum and 5% newborn calf serum, as described
previously(19) . Within 24 h after passage, cells were
prelabeled with 5 nCi/ml [C]dThd (DuPont NEN)
for 1 week, the labeled medium was replaced with fresh medium, and the
cells were grown for an additional week to become confluent. Prior to
UV damage, the cells were treated for 45 min with 2 mM hydroxyurea to arrest the low level (
0.5%) of replicating
cells in these populations (e.g. see Ref. 20). The medium was
then removed, and cells were washed with warm phosphate-buffered saline
(140 mM NaCl, 2.6 mM KCl, 6.8 mM
Na
HPO
, 1.5 mM
KH
PO
) and irradiated in Petri dishes with
254-nm light in a (desktop) hood containing a single low pressure
mercury lamp (Sylvania, model G 30T8), as described
previously(19, 21) . Irradiation was carried out in a
warm (37 °C) room and the UV flux measured with a Spectroline
DM-254N UV meter (Spectronics Corporation, Westbury, NY). Cells were
either harvested immediately after irradiation or incubated for varying
repair times (see text).
Nuclei Isolation
After the various repair times,
cells were harvested and nuclei isolated as described by Smerdon et
al. (19). Nuclei were digested in B3 buffer (10 mM
Tris-HCl, pH 8, 0.1 mM CaCl, 0.25 mM sucrose) with micrococcal nuclease (at 1 unit/10
nuclei) for 15 min at 37 °C. The 146-bp core DNA was
electrophoretically isolated as described
previously(22, 23) . Briefly, after digestion of nuclei
with micrococcal nuclease, the mixture of oligo- and mononucleosomes
was digested with proteinase K and core DNA isolated on 2.5%
preparative agarose gels. The 146-bp core DNA band was excised,
electroeluted into TBE (90 mM Tris-HCl, pH 8.3, 90 mM
boric acid, 2.5 mM EDTA) using an electroelution device
(Elutrap, Schleicher & Schuell), concentrated with isobutyl
alcohol, and cleaned on a Sephadex G-50 (Sigma) spin column. Genomic
DNA was prepared according to Marzluff and Huang (24) (see also
Ref. 25). After lysis of cells and proteinase K digestion, the DNA was
phenol/chloroform-extracted, ethanol-precipitated, and digested with PstI. Cesium chloride gradient centrifugation for 17 h at
55,000 rpm was used to separate RNA from the DNA. The DNA fractions
were pooled, dialyzed extensively, concentrated with isobutyl alcohol,
and further purified on a Sephadex G-50 (Sigma) spin column.
Concentrations of core and genomic DNA were obtained from absorbance
scans.
Radioimmunoassay
Details of the radioimmunoassay
(RIA) have been published(26, 27) . DNA from Clostridium perfringens (Sigma) was nick-translated with
[-
P]dCTP (Amersham Corp.) to a
specific activity of 5-10
10
cpm/µg. The
labeled DNA was irradiated in water at a fluence rate of 14
watts/m
measured at 254 nm for a total fluence of 30
kJ/m
. About 10 pg (5000-10,000 cpm) of UV-irradiated DNA
competed with sample DNA for binding to antiserum. Anti-(6-4)PD
or anti-CPD sera was added to 10 mM Tris-HCl (pH 7.8), 150
mM NaCl, and 1 mM EDTA containing 0.15% gelatin (Type
III, Sigma). The specificities of these antisera for)PDs and cis-syn cyclobutane dimers have been demonstrated previously
(see Ref. 27, and references therein). After overnight incubation at 4
°C, goat anti-rabbit IgG (Calbiochem) and carrier
-globulin
(Calbiochem) were added and incubated for 2-3 days at 4 °C to
form a precipitable immune complex. The immune pellet was collected by
centrifugation, dissolved in tissue solubilizer (Amersham Corp.), and
assayed for radioactivity in a Packard liquid scintillation counter.
Calculations of photoproduct yield were obtained from standard curves
in the RIA generated from salmon sperm DNA (Sigma) irradiated with
increasing fluences of 254-nm UV light. The percent inhibition of
sample DNA was extrapolated through a linear regression of the standard
and yields of photoproduct calculated based on induction rates of 2.45
CPD/10
daltons/J/m
/s and 0.56)PD/10
daltons/J/m
/s(8) .
Figure 1:
Purification of core and chromatosome
DNA from human cells. Nuclei were isolated from confluent human
fibroblasts (AG1518 cells) and digested with micrococcal nuclease until
about 20% of the DNA was rendered acid-soluble, according to Lan and
Smerdon (22). Core and chromatosome DNA was separated on 2.5%
preparative agarose gels and electroeluted. Samples are: total DNA
after micrococcal nuclease digestion (lane1); 146-bp
core DNA after electroelution (lane2); and
(predominately) 166-bp chromatosome DNA after electroelution (lane3). Markers (laneM) are from a HpaII digest of plasmid pBluescript II KS, a
2961-bp plasmid from Stratagene (Menasha,
WI).
Samples were then subjected
to RIA, using antibodies to either (6-4)PDs or CPDs. The
inhibition of antibody binding to irradiated, P-labeled
standards (see ``Materials and Methods'') was determined for
samples at each UV dose. The amounts of DNA required to give 50%
inhibition of antibody binding, compared with the known amounts of CPDs
and)PDs in the standards, were used to calculate the numbers of
photoproducts/µg of core DNA. As shown in Fig. 2, the
overall yield of)PDs in core DNA is about 30% that of CPDs. We chose a
dose of 40 J/m
for repair experiments to allow accurate
measurement of)PDs, without any observable death of the confluent
cells.
Figure 2:
UV dose
response of nucleosome core DNA in human cells. Confluent AG1518 cells
were irradiated at varying UV doses with (primarily) 254-nm light.
Nucleosome core DNA (146 bp) was isolated from micrococcal
nuclease-digested nuclei (see Fig. 1) immediately following
irradiation. Photoproducts were measured in purified core DNA by RIA
using antibodies specific to CPDs () and (6-4)PDs (
).
Yields were calculated from the 50% inhibition of binding to
UV-irradiated standards at each UV dose (see ``Materials and
Methods'').
The level of CPDs and)PDs remaining following different
repair times (0-24 h) was then measured by performing RIAs on
samples isolated following different incubation times after UV
irradiation. Surprisingly, about 75% of the)PDs are removed from core
DNA in only 2 h, and over 90% are removed in 24 h (Fig. 3, closedsymbols). This is in marked contrast to the
slow removal of CPDs over the same time period (Fig. 3, opensymbols). To test if the slower rate of CPD
removal is partly due to the higher yield of CPD lesions in core DNA (Fig. 2), we reduced the UV dose to 10 J/m,
where the CPD level is less than (or similar to) that of)PDs at 40
J/m
. Once again, slow removal of CPDs from core DNA was
observed (Fig. 3, opentriangles) and
was similar to that following 40 J/m
.
Figure 3:
Repair of CPD and (6-4)PD
photoproducts in nucleosome core DNA of human cells. Confluent AG1518
cells were irradiated with 40 or 10 J/m and allowed to
repair for varying times. Preparations of nucleosome core DNA and RIAs
were performed at each repair time (see Figs. 1 and 2), and the
fraction of CPDs and (6-4)PDs remaining at each time was
determined. Data are for CPDs (
,
) and (6-4)PDs
(
,
) remaining after 40 J/m
and CPDs remaining
after 10 J/m
(
). Different symbols for 40 J/m
data represent values for either the mean of two or three
experiments (circles) or one experiment (squares) at
each time point.
We also examined
removal of CPDs and)PDs from chromatosome DNA (166 bp), isolated as
above (see Fig. 1). Since the CPD yield is greatest near
the ends of core DNA (28) and this DNA is less tightly bound to
histones, we wondered if the repair kinetics would be different from
repair of CPDs in core DNA of human cells. However, as with core DNA,
we observed a much slower rate of removal of CPDs within chromatosome
DNA and a much faster rate of removal of)PDs (data not shown). The
rates of removal of each photoproduct were very similar to those
observed for core DNA (Fig. 3).
10 times faster.
During this time, most repair of CPDs occurs in transcriptionally
active regions of chromatin (reviewed in Refs. 30 and 31). Thus, the
vast majority of)PDs is removed from genomic DNA within the time period
that actively transcribed genes are repaired (i.e. <20% of
genomic DNA). Our results demonstrate that this includes the highly
``nuclease-protected'' regions of nucleosome cores in human
chromatin.
Table: CPD and (6-4)PD Yield in Nucleosome DNA
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.