©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Repair of UV-induced) Photoproducts in Nucleosome Core DNA (*)

Christine Suquet , David L. Mitchell (1), Michael J. Smerdon

From the (1)Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-4660 and the University of Texas, M. D. Anderson Cancer Center, Smithville, Texas 78957

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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. 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.


INTRODUCTION

There are two major classes of stable photoproducts induced in DNA by ultraviolet (UV) radiation, namely cis-syn cyclobutane pyrimidine dimers (CPDs)()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).

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.


MATERIALS AND METHODS

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 NaHPO, 1.5 mM KHPO) 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) .


RESULTS

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.


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).

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.


DISCUSSION

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 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.

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.

  
Table: CPD and (6-4)PD Yield in Nucleosome DNA



FOOTNOTES

*
This study was supported by National Institutes of Health Grants ES02614 (to M. J. S.) and ES05914 (to D. L. M.) 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. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: CPDs, cis-syn cyclobutane pyrimidine dimers; (6-4)PDs, pyrimidine-pyrimidone (6-4) dimers; bp, base pair; RIA, radioimmunoassay; NER, nucleotide excision repair.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.