©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Implication of Mammalian Ribosomal Protein S3 in the Processing of DNA Damage (*)

Joon Kim (1)(§), Leda S. Chubatsu (1)(¶), Arie Admon (1)(**), Joachim Stahl (2), Robert Fellous (1)(§§), Stuart Linn (1)(¶¶)

From the (1) Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, California 94720-3202 and the (2) Max Delbrück Centrum für Molekulare Medizin, Robert Rössle Strasse 10, 13125 Berlin-Buch, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S.(1978) Nucleic Acids Res. 5, 951-960). We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity. This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer. The nuclease activities have been purified from mouse cells to yield a peptide of M = 32,000, whose sequence indicates identity with ribosomal protein S3. The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III. Moreover, the protein expressed in E. coli and its activities cross-react with the rat protein antibody. Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding. The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed.


INTRODUCTION

UV radiation is probably the most extensively used model system for investigating the biological consequences of DNA damage. When DNA is exposed to radiation at wavelengths near its absorption maximum of 260 nm, a large number of damages are formed, the most prevalent being cyclobutane pyrimidine dimers (Loeber and Kittler, 1977). These dimers are chemically stable and are corrected either by nucleotide excision repair or in some cell types by enzymatic photo reversal (Boyce and Howard-Flanders, 1964).

Xeroderma pigmentosum (XP)() is a rare, autosomal recessive disease that is clinically characterized by hypersensitivity to ultraviolet radiation and a large increase in the frequency of sunlight-induced skin carcinomas or malignant melanomas (Kraemer et al., 1984) and in some cases by UV-induced ocular lesions and progressive neurologic degeneration. Cells cultured from XP patients exhibit enhanced sensitivity to killing by UV light (Kraemer et al., 1976; Andrews et al., 1978) and display reduced levels of UV-induced DNA repair synthesis (Cleaver, 1968) as well as inefficient removal of a wide range of chemically induced bulky DNA damages (Amacher et al.,1977; Galloway et al.,1994). Based upon restoration of UV-induced repair synthesis following cell fusion, eight complementation groups have been defined: seven classical groups (A-G) that are abnormal in excision repair capability (de Weerd-Kastelein et al., 1972; Hoeijmakers, 1993) and a variant group that exhibits normal levels of excision repair but has impaired post replication repair (Lehmann et al., 1975).

Kuhnlein et al.(1978) found that XP-D cells, and only cells from that complementation group, lack or have altered an AP endonuclease, designated AP endo I. This abnormality apparently manifests itself through a higher than normal ability of XP-D cells to host cell-reactivated depurinated SV40 DNA (Kudrna et al. 1979). (As expected, on the other hand, in the same study, XP-D cells displayed inefficient host cell reactivation of UV-irradiated SV40 DNA.) The abnormality of the AP endonuclease activity in XP-D cells is peculiar, however, since XP-D cells are known to carry mutations in the gene for a DNA helicase activity (Weber et al., 1990). We now report that this AP endonuclease activity is inseparable from activities acting upon UV-irradiated DNA, one of which cleaves a phosphodiester bond within a cyclobutane pyrimidine dimer. Moreover, these activities are apparently associated with ribosomal protein S3, a protein located on the external surface of the 40 S ribosomal subunit, which assembles onto preribosomal particles in the nucleus (Hadjiolov, 1985). Possible roles of this ribosomal protein in processing DNA damage are discussed.


EXPERIMENTAL PROCEDURES

Materials

Murine plasmacytoma cell strain MPC-11 was obtained from Dr. M. Koshland (University of California, Berkeley). Human normal fibroblast strain F65 was obtained from the Naval Biomedical Research Laboratory (Oakland, CA) and XP fibroblast strain GM5424 (complementation group D) was from the National Institute of General Medical Sciences Human Genetic Mutant Cell Repository (Camden, NJ). Normal (GM 3714) and XP group D (GM 2485-A) human lymphoblasts were from Dr. T. Lindahl (Imperial Cancer Research Fund, London, United Kingdom). Supercoiled PM2 phage [thymidine-H]DNA (5000-16000 cpm/nmol) was purified as described (Kuhnlein et al., 1976). U15A RNA was synthesized using SP6 RNA polymerase and plasmid pS3 (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) (Tycowski et al.,1993) linearized with NsiI as described by Melton et al.(1984). Phosphocellulose was from Sigma, DE-52 DEAE-cellulose was from Whatman, and BA85S597 0.45-mm nitrocellulose membrane filters were from Schleicher and Schuell. [methyl-H]Thymidine was from Amersham.

Antibodies S3 71/83 and S3 67/67 were isolated by immunoabsorption from rabbit antisera prepared against purified rat liver ribosomal protein S3. As a control, immunoabsorption-purified antibodies raised in rabbits against rat ribosomal protein S26 (S26-67/8) were used. These antibodies are described by Lutsch et al.(1990).

Escherichia coli endonuclease III was prepared and assayed according to Gates and Linn(1977), T4 UV endonuclease was prepared according to Friedberg and King(1971), and HeLa AP endonuclease II was prepared according to Kane and Linn(1981). E. coli DNA polymerase I was purchased from Boehringer Mannheim or from Life Technologies, Inc. Uracil DNA glycosylase was prepared as described by Gates and Linn(1977). The T4 endonuclease (Ser) construct was originally obtained from Drs. J. K. de Riel and K. Valerie (VA Commonwealth University). It has a W128S substitution due to substitution of an AGT for a TGG codon, a substitution that also generates a unique SpeI site in the ptac-denV plasmid construct.

Cell Culture

MPC-11 cells were split 1:2 daily in suspension medium containing 50% Dulbecco's modified Eagle's medium, 50% RPMI 1640 medium (Life Technologies, Inc.), 20% heat-inactivated horse serum, 2 mML-glutamine, 100 units/ml each of penicillin and streptomycin, 0.02 M Hepes (pH 7.2), and 50 µM 2-mercaptoethanol. F-65 diploid human fibroblasts were cultured in a humidified 5% CO incubator at 37 °C in 150-cm flasks. XP fibroblasts were grown and maintained in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. GM 3714 normal lymphoblasts and GM 2485A XP-D lymphoblasts were grown in RPMI 1640 medium using roller bottles in the dark.

Preparation of Damaged DNAs

Depurination of PM2 DNA was carried out at 70 °C in 10 mM sodium citrate (pH 4.5), 100 mM NaCl for 10 min so as to generate 1.5 AP sites per DNA duplex. PM2 DNA was UV-irradiated in 10 mM Tris-HCl (pH 7.5) and 0.02% glycerol at 2.5 J/m/s for the times indicated with a Westinghouse germicidal lamp (G15T8). As a comparison, a total dose of 525 J/m produces approximately 2 sites sensitive to E. coli endonuclease III per PM2 DNA duplex (Demple and Linn, 1982), whereas 23 J/m produces roughly 2 cyclobutane pyrimidine dimers per molecule (Snapka and Linn, 1981).

Endonuclease Assays

A filter-binding assay with nitrocellulose filters and covalently closed, circular (form I) PM2 DNA was used to monitor endonuclease activity. Standard reaction mixtures (100 µl) for UV endonuclease activity contained 40 mM Tris-HCl (pH 8.0), 70 mM KCl, 0.01% Triton X-100, 3 mM EDTA, 10 mM 2-mercaptoethanol, and 20 µM (nucleotide residues) UV-irradiated PM2 [H]DNA. AP endonuclease assays were similar except that depurinated PM2 [H]DNA was substrate. Reactions were incubated for the indicated times at 37 °C and then terminated by the addition of 100 µl of 0.3 M KHPO (pH 12.4), which partially denatures the PM2 DNA. To detect total DNA by total denaturation, 100 µl of 0.3 M KHPO (pH 13.2) was added. A sample of the reaction mixture was also spotted directly onto the filter for determining the total amount of DNA present. After 5 min, the samples were neutralized with 50 µl of 1 M KHPO (pH 4.0), and then 100 µl of 5 M NaCl was added. This treatment results in the renaturation only of form I DNA. Each sample was finally diluted to 3 ml with 50 mM Tris-HCl (pH 8.2), 1 M NaCl and filtered through a Schleicher and Schuell nitrocellulose filter to selectively retain the denatured DNA. The filters had been previously equilibrated with the same buffer. After being washed with 5 ml of 50 mM Tris-HCl (pH 8.2), 1 M NaCl and then with 5 ml of 0.3 M NaCl, 0.02 M sodium citrate, the filters were dried and counted by liquid scintillation.

The average number of nicks introduced per PM2 genome was calculated from the percentage of total PM2 [H]DNA bound to each filter assuming a Poisson distribution of endonucleolytic sites (Kuhnlein et al., 1976). 1 unit of rpS3 endonuclease activity specifically introduces 1 fmol of nicks into depurinated or heavily UV-irradiated DNA per min at 37 °C. 1 unit of T4 UV endonuclease produces 1 fmol of alkali-labile sites in DNA containing cyclobutane pyrimidine dimers as measured in .

Separation of Three UV Endonucleases from MPC-11 Cells

96 liters of culture (1.1 10 cells) in logarithmic growth were harvested and resuspended in a final volume of 200 ml of 10 mM Tris-HCl (pH 8.0), 0.7 M KCl, 1 mM EDTA, 5.0% sucrose, and 10 mM 2-mercaptoethanol, and then the suspension was immediately sonicated five times for 15 s with a Branson sonifier and a large probe. After adjustment to a final concentration of 0.3 M KCl with 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol, the extract was further sonicated four times for 15 s, then gently stirred for 2 h at 0 °C and centrifuged twice at 12,000 rpm for 20 min in a Sorvall GSA rotor at 4 °C.

The supernatant was loaded onto a DE-52 DEAE-cellulose column (5 16 cm), which had been equilibrated with 0.4 M NaCl, 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 mM 2-mercaptoethanol to remove nucleic acids, and then the column was eluted with the same buffer at a flow rate of 100 ml/h. Flow-through fractions (400 ml), which had significant UV absorbance at 280 nm, were pooled and dialyzed against 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, and 10 mM 2-mercaptoethanol (buffer A).

The dialysate was loaded onto a 1.2-liter phosphocellulose column, which was equilibrated with buffer A at a flow rate of 50 ml/h. It was washed with the same buffer, and flow-through fractions, which contain UV endonuclease III, were collected. UV endonucleases I and II were eluted from this column with a 4-liter linear gradient from 0 to 1.2 M KCl in buffer A (see Kim and Linn(1989)).

Further Purification of UV Endonuclease III from MPC-11 Cells

96 liters of culture (1.1 10 cells) in logarithmic growth were harvested 8 liters at a time, washed once with PBS, frozen as a pellet in liquid nitrogen, and stored at -70 °C for up to 30 days. The total collection of cells was thawed and resuspended in a final volume of 200 ml of 10 mM Tris-HCl (pH 8.0), 0.7 M KCl, 1 mM EDTA, 5.0% sucrose, and 10 mM 2-mercaptoethanol, and then the suspension was immediately sonicated five times for 15 s with a Branson sonifier and a large probe. After adjustment to a final concentration of 0.3 M KCl with 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol (buffer B), the extract was further sonicated four times for 15 s, then gently stirred for 2 h at 0 °C and centrifuged twice at 12,000 rpm for 20 min with a Sorvall GSA rotor at 4 °C.

The supernatant was loaded onto a DEAE-cellulose column (5 16 cm), which had been equilibrated with 0.4 M NaCl, 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 mM 2-mercaptoethanol to remove nucleic acids, and then the column was eluted with the same buffer at a flow rate of 100 ml/h. Flow-through fractions, which had significant UV absorbance at 280 nm, were dialyzed against buffer B and loaded at a flow rate of 50 ml/h onto a 1.2-liter phosphocellulose column, which was equilibrated with buffer B. The column was washed with the same buffer, and fractions that contained UV endonuclease III were collected (UV endonucleases I and II could be also eluted from this column with a 4-liter linear gradient from 0 to 1.2 M KCl in buffer B (see Fig. 1 )).


Figure 1: Phosphocellulose chromatography of murine UV endonucleases. Preparation and chromatography of the extract from MPC-11 cells are described under ``Experimental Procedures.'' The characterization of the activities in peaks I and II was previously described by Kim and Linn (1989).



The phosphocellulose fraction was loaded onto a 400-ml second DEAE-cellulose column, which had been equilibrated with buffer B. The column was washed with buffer B and then eluted with a 1.2-liter linear gradient from 5 to 200 mM potassium phosphate (pH 8.1) in 0.01% Triton X-100, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol. Enzyme eluted at 90 mM potassium phosphate, and active fractions were pooled.

To the dialysate (164 ml) was added 26.8 g of ammonium sulfate with constant stirring on ice; then, after 10 min, it was centrifuged for 15 min at 20,000 g at 4 °C. To the supernatant was added 40 g of ammonium sulfate, and the precipitate was collected as above and dissolved in buffer B to a final volume of 8 ml.

The above material was loaded onto a Sephacryl S-200 column (140 ml, bed volume), which had been equilibrated with 25 mM potassium phosphate, 0.01% Triton X-100, and 10 mM 2-mercaptoethanol. Activity separated into 2 peaks, which eluted after 64 and 106 ml. The second peak was dialyzed against 5 mM potassium phosphate (pH 8.1), 0.01% Triton X-100, and 10 mM 2-mercaptoethanol (buffer C). It was subsequently found that all of the activity elutes in the position of the second peak if 0.7 M ammonium sulfate is included in the elution buffer.

A 0.5-ml hydroxyapatite column was equilibrated with buffer C, and the dialysate was applied to the column; then, the column was washed with the same buffer and eluted with a linear gradient from 5 to 300 mM potassium phosphate (pH 8.1) in buffer C and collected in 0.5-ml fractions. UV endonuclease III activity eluted at 70 mM potassium phosphate, and active fractions were pooled and dialyzed against buffer C.

Quantitation of UV Endonuclease III Activity from Normal and XP-D Human Cells

Log phase normal F65 fibroblast cells (40 T-150 flasks) or XP-D fibroblast cells (15 T-150 flasks) were harvested by scraping, rinsed with PBS, resuspended, sonicated, and centrifuged as described for the purification of the murine enzymes (Kim and Linn, 1989). The supernatant was collected and loaded onto a 4-ml DEAE-cellulose column equilibrated with 0.4 M NaCl, 40 mM Tris-HCl (pH 8.0), 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 0.005% Triton X-100 to remove nucleic acids. Flow-through fractions (39 ml for normal and 15 ml for XP-D cells) from the DEAE-cellulose column, which had significant UV absorbance at 280 nm, were pooled and dialyzed against 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.005% Triton X-100 (buffer B) and loaded onto a phosphocellulose column (15 ml for normal and 8 ml for XP-D cells), which had been equilibrated with buffer B and then washed with the same buffer. UV endonuclease III activity flowed through the column. A steep gradient from 0 to 1.2 M KCl in buffer B was applied to elute UV endonucleases I plus II for comparison.

GM 3714 normal lymphoblast cells (5.6 liters of culture, 6.7 10 cells), GM 2485A XP-D lymphoblast cells (5.6 liters, 3.7 10 cells), or MPC-11 cells (4.8 liters, 3.7 10 cells) were harvested, rinsed with PBS, and then resuspended in 5 volumes of TE sucrose buffer (10 mM Tris (pH 7.5), 1.0 mM EDTA, and 0.25 M sucrose). The cells were lysed by homogenization in a Waring blender three times for 10 s at maximum power, and then the blender was rinsed with an additional 5 ml of TE sucrose buffer. Nuclei and cell debris were pelleted by centrifugation (Anderson and Friedberg, 1980), and then the supernatant was collected by centrifugation in a Sorvall SS34 rotor at 12,500 rpm for 30 min to remove mitochondria and finally dialyzed against 40 mM Tris (pH 8.0), 10 mM KCl, 0.05% Triton, and 10 mM 2-mercaptoethanol. Dialysates containing about 10 mg of protein from normal and XP-D lymphoblast cells were each loaded onto 5-ml phosphocellulose columns, which were equilibrated with 40 mM Tris (pH 8.0), 10 mM KCl, 0.05% Triton, and 10 mM 2-mercaptoethanol. Flow-through fractions were collected, and, as a reference, UV endonucleases I and II were eluted from the columns with a buffer containing 1.2 M KCl, 40 mM Tris (8.0), 10 mM KCl, 0.05% Triton, and 10 mM 2-mercaptoethanol.

Incision of PM2 DNA and DNA Synthesis Reactions

Reactions for incision (100 µl) contained 40 mM Tris-HCl (pH 8.0), 3 mM EDTA, 0.01% Triton X-100, 70 mM KCl, 5 mM 2-mercaptoethanol, 40 µM depurinated PM2 [H]DNA, and endonucleases as indicated. Incubation was at 37 °C, and the reactions were stopped by heating for 5 min at 70 °C. Duplicate aliquots of 10 µl were removed to determine the extent of endonuclease incision. Reactions for DNA synthesis (200 µl) contained 70 mM potassium phosphate (pH 7.5), 9 mM 2-mercaptoethanol, 7 mM MgCl, 90 µM each of dATP, dGTP, and dCTP, 85 µCi of [-P]dTTP (500 cpm/pmol), 2.6 nmol of the incised PM2 [H]DNA, and E. coli DNA polymerase I. Incubation was at 37 °C, and aliquots of 50 µl were removed at 10-min intervals, chilled on ice, and then mixed with 200 µl of 0.1 M sodium pyrophosphate and 50 µl of 5 mg/ml bovine serum albumin prior to precipitation with 0.7 ml of 10% trichloroacetic acid. The precipitates were filtered onto Whatman GF/C filters, washed with 50 ml of 1 N HCl, 0.1 M sodium pyrophosphate and then dried with 5 ml of 95% ethanol. Radioactivity retained on the filter was measured by double isotope counting using liquid scintillation with Betamax (West Chem) fluor.

Identification of the Product of Combined Reactions with Class II AP Endonucleases

[P,uracil-H]Poly(dA-dT) was prepared, and uracil DNA glycosylase was assayed as previously described (Kim and Linn, 1988). 1 unit of the glycosylase releases 1 pmol of uracil per min from phage PBS2 DNA at 37 °C. AP endonuclease activity was measured as described above; 1 unit of activity produces 1 fmol of nicks specifically into AP DNA per min at 37 °C. Reaction mixtures containing 50 µM [uracil-H]poly(dA-dT) were incubated for 20 min at 37 °C to produce approximately 1 pmol of AP sites. After enzyme inactivation for 5 min at 70 °C, the reactions were placed on ice and brought to 40 mM Tris-HCl (pH 8.0), 70 mM KCl, 0.01% Triton X-100, and 3 mM EDTA, and AP endonucleases were added. After the enzyme reaction, the products were spotted onto Whatman 3MM chromatographic paper (27 45 cm) and chromatographed as previously described (Kim and Linn, 1988).

Subcloning, Overexpression, and Purification of Rat Ribosomal Protein S3 from E. coli

NdeI and BamHI restriction sites were introduced at the beginning and the end of the rpS3 cDNA, respectively, by polymerase chain reaction mutagenesis from pGEM2-S3-15 (Chan et al., 1990). The polymerase chain reaction fragment was subcloned into the T7 RNA polymerase expression vector, pT7-7 (Tabor and Richardson, 1985) using the NdeI and BamHI restriction sites to produce pT7-7S3. Then, pT7-7S3 was introduced into E. coli strain BL21plysS.

10 liters of cells were grown to a density of A = 0.5 and then induced by the presence of isopropyl-1-thio--D-galactopyranoside at a final concentration of 1 mM for 4-5 h at 37 °C. The bacteria were pelleted by centrifugation and resuspended in buffer C (20 mM Tris-HCl (pH 7.5), 10% glycerol, and 1 mM dithiothreitol) with 0.5 M KCl and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin), sonicated 6 30 s with a Branson sonicator and the large probe on ice, and then centrifuged for 20 min at 12,000 g at 4 °C. The supernatant was loaded onto a DEAE-cellulose column equilibrated with buffer C plus 0.5 M KCl. The flow-through was dialyzed overnight against buffer C plus 0.1 M KCl and loaded onto an S-Sepharose column. Ribosomal protein S3 was eluted with a gradient of 0.1-1 M KCl in buffer C. The fractions containing rpS3 (0.50-0.55 M KCl) were pooled, concentrated 20-fold using an Amicon apparatus, and loaded onto a fast protein liquid chromatography-Superose 12 HR 10/30 column equilibrated with buffer C plus 0.1 M KCl. The column was eluted with the same buffer. Fractions of 0.3 ml each were collected, and the activity eluted after 14 ml.


RESULTS

Purification of UV Endonuclease III Activity and Its Absence or Chromatographic Abnormality in XP Group D Cells

When extracts from cultured murine or human cells (data not shown) are passed through phosphocellulose, three peaks of activity, which nick heavily UV-irradiated DNA, are observed (Fig. 1). The two peaks of activity which bind to phosphocellulose, and UV endonucleases I and II, were purified and characterized previously (Kim and Linn, 1989). The flow-through activity, which we called UV endonuclease III, is the subject of this report. We have now purified UV endonuclease III by chromatography upon DEAE-cellulose, phosphocellulose,sephacryl S-200, hydroxyapatite, heparin-agarose, and S-Sepharose followed by sucrose gradient sedimentation. The final preparation has a specific activity of roughly 33,000, a value that is comparable with 38,000 and 29,000 obtained for UV endonucleases I and II, respectively, from the same cells (Kim and Linn, 1989). Throughout the purification procedure, the activity was inseparable from the AP endonuclease activity present in the phosphocellulose flow-through fraction,() previously designated as AP endonuclease I (Kuhnlein et al., 1976, 1978).

Since the phosphocellulose flow-through fraction from XP-D cells was observed specifically to lack AP endonuclease I activity (Kuhnlein et al., 1978), phosphocellulose flow-through fractions from normal and XP-D human fibroblasts and lymphoblasts and from normal rodent MPC-11 cells were compared for their UV endonuclease III activity. Indeed, this activity from XP-D cells was significantly lower or absent compared with that from the corresponding normal cells (). The content of UV endonucleases I and II activities, however, did not differ greatly between the XP-D and the corresponding normal cells (). This concurrent reduction suggested that the two activities could be associated with the same protein.

UV Endonuclease III Substrate Specificity

Characterization of the AP Endonuclease Activity

To characterize the murine and human AP endonuclease I activities, depurinated PM2 DNA was incised with either the human or murine enzyme. Nicks were produced that did not support DNA synthesis with E. coli DNA polymerase I (Fig. 2). These results indicate that these enzymes leave a baseless sugar, a sugar phosphate, or a phosphomonoester at the 3` terminus. To distinguish among these possible cleavage products at the 3` terminus, we examined the products formed by the combined action of the AP endonuclease activity of human or murine UV endonuclease III and the major class II AP endonuclease from HeLa cells (see Kim and Linn (1988)). In both cases, the product was an unsaturated sugar residue, indicating that UV endonuclease III, like UV endonucleases I and II, mediates phosphodiester bond cleavage via a class I -elimination process ().


Figure 2: Mechanism of action of AP endonuclease activities of human and murine UV endonuclease III. Reactions at 37 °C as described under ``Experimental Procedures'' utilized 5 units of E. coli endonuclease III for 15 min, 5 units of AP endonuclease II for 15 min, 1 unit of human UV endonuclease III for 40 min, and 0.1 unit of DNA polymerase I (panelA) or 4 units of murine UV endonuclease I (a class I -lyase enzyme) for 40 min, 5 units of AP endonuclease II for 40 min, 4 units of murine UV endonuclease III for 40 min, and 0.18 unit of DNA polymerase I (panelB). The PM2 DNA was first incised by the endonucleases and then used as substrate for DNA synthesis with E. coli DNA polymerase I. The number of incisions per duplex DNA molecule, which had been made by each endonuclease, is given in parentheses. The UV endonuclease III used in these experiments were hydroxyapatite fractions.



UV Endonuclease III Interacts with Cyclobutane Pyrimidine Dimers

Since UV endonuclease III activity appeared to be weak or absent in fractions from XP-D cells and since XP-D cells are defective in excising cyclobutane pyrimidine dimers (Galloway et al., 1994), interaction of the enzyme with DNA containing cyclobutane pyrimidine dimers was explored. To generate approximately 4 cyclobutane pyrimidine dimers (and roughly 1 pyrimidine-pyrimidone (6-4) photo product) per covalently closed, circular phage PM2 DNA molecule without generating a significant number of other base or sugar damages (Demple and Linn, 1982), DNA was irradiated at a dose of 46 J/m. This DNA was compared as a substrate to PM2 DNA, which had received a dose of 525 J/m so as to generate DNA molecules containing not only cyclobutane pyrimidine dimers but also a significant frequency of hydrated pyrimidines, AP sites, cross-links, and a number of other damages. When the more lightly irradiated DNA was treated with UV endonuclease III, no conversion of form I to form II DNA was detected; however, DNA receiving 525 J/m of irradiation was converted to form II by the enzyme (I). Hence, the enzyme puts observable nicks into DNA containing damages brought about by heavy irradiation but not into DNA containing predominantly cyclobutane pyrimidine dimers and some) photo products as the damage products.

Since this activity appeared to be reduced in XP-D cells and since XP-D cells have a reduced ability to process cyclobutane pyrimidine dimers, we wondered whether the enzyme might have a more subtle interaction with pyrimidine dimers. Weinfeld et al.( (6) reported that excised cyclobutane pyrimidine dimers isolated as dinucleoside monophosphates from the growth medium of UV-irradiated human fibroblasts released free thymidine and thymidine monophosphate after reversal of the dimerization with high doses of UV light. This result was taken to imply that the photo-liberated thymidine and thymidine monophosphate had been attached solely by the cyclobutane ring of the dimer, i.e. that there had been a cleavage of an intradimer phosphodiester linkage during the in vivo processing of DNA cyclobutane pyrimidine dimers. To test whether UV endonuclease III might catalyze such a phosphodiester bond cleavage within the pyrimidine dimer, the consequence of sequential reactions of murine UV endonuclease III followed by the pyrimidine-dimer glycosylase activity associated with T4 endonuclease V was studied (see Fig. 3). In this experiment, a mutant form (Nakabeppu et al., 1982) of the T4 endonuclease, T4 endonuclease (ser), that lacks most of the AP endonuclease activity of the enzyme but retains the DNA glycosylase activity was used.


Figure 3: Proposed sequential reactions of UV endonuclease III and the cyclobutane pyrimidine dimer glycosylase activity of T4 UV endonuclease (Ser). If UV endonuclease III were to cleave a phosphodiester bond within a cyclobutane dimer contained in form I PM2 DNA, a nick would not be observed as it would be masked by the pyrimidine-pyrimidine linkage. Breakage of the 5`-pyrimidine glycosylic bond with phage T4 endonuclease V would expose the nick, however, and give rise to form II DNA.



PM2 DNA was irradiated with a dose of 23 J/m to yield roughly 2 cyclobutane pyrimidine dimers per molecule, and then the sequential reactions shown schematically in Fig. 3were carried out (, Experiment I). The UV endonuclease III alone produced neither observable nicks nor alkali-labile sites (in particular, alkali-labile AP sites) in this lightly UV-irradiated DNA. Hence, UV endonuclease III neither nicks DNA adjacent to pyrimidine dimers nor does it form AP sites or other alkali-labile sites.

The amount of mutant T4 enzyme used in this experiment, on the other hand, while producing no nicks, produced a number of alkali-labile sites equal to roughly half of the pyrimidine dimers present (alkali-labile sites are presumably sites at cyclobutane pyrimidine dimers, which have been subject to the DNA glycosylase activity of the T4 enzyme but not to phosphodiester bond cleavage) (see Fig. 3). The ordered reaction of the mammalian enzyme followed by the phage DNA glycosylase activity, however, resulted in the detection of a number of nicks equal to the number of cyclobutane pyrimidine dimers present in the DNA substrate. In addition, no alkali-labile sites were present in this DNA, which had been treated with both enzymes. Hence, the combination of activities ultimately generated one nick for each pyrimidine dimer present and left no additional substrate for the T4 enzyme to convert to alkali-labile sites. Notice that this experiment demonstrates that the T4 enzyme acts more efficiently upon the UV-irradiated DNA, which had been exposed to the UV endonuclease III than upon the DNA prior to exposure to the mammalian protein. In other experiments utilizing more of the enzyme, the T4 enzyme alone could convert all pyrimidine dimers present to alkali-labile sites. In conclusion, the UV endonuclease III appears to be able to cleave a phosphodiester bond between the dimerized pyrimidine nucleoside residues, but these cleavages are observable only after exposure to the T4 DNA glycosylase (see Fig. 3 ).

The above experiment utilized nitrocellulose filter binding to monitor DNA nicking. To verify that the filter binding truly reflected cleavage of the DNA backbone, unirradiated and irradiated PM2 DNAs were treated with the combination of enzymes and then sedimented through denaturing, alkaline sucrose gradients. DNA, which was irradiated and treated with both enzymes, was indeed converted from form I to form II and smaller DNA species (Fig. 4). Significantly, irradiated DNA treated with either one of the enzymes alone did not exhibit any altered sedimendation pattern. Similar results were obtained when DNA nicking was monitored by gel electrophoresis (data not shown).


Figure 4: Nicking of UV-irradiated PM2 DNA by sequential reactions with UV endonuclease III and T4 UV endonuclease (Ser) as demonstrated by alkaline sucrose gradient sedimentation. Reactions were carried out as in Table IV with H-labeled PM2 DNA irradiated with 23 J/m where indicated and then sedimented through 5-25% gradients of sucrose containing 0.25 M NaOH, 0.9 M NaCl, and 5 mM EDTA at 20 °C for 75 min at 43,000 rpm in a Beckman SW50.1 rotor. The gradients were collected from the tube bottoms and radioactivity determined. A, sequential enzyme reactions where indicated. B, T4 enzyme only.



Another technique for monitoring a phosphodiester cleavage within a cyclobutane pyrimidine dimer would be to treat with photolyase, an enzyme that reverses the pyrimidine dimerization to regenerate normal pyrimidine bases on the DNA. When this scheme was tried, erratic results were obtained. The photolyase did not act well on the product of the UV endonuclease III (in contrast to the T4 endonuclease V, which preferred it). This weak activity of the photolyase may have been due to the presence of the nick within the dimer in a supercoiled substrate and/or the particular phosphodiester cleavage (3` or 5`) within the dimer. In particular, the photolyase appeared to act, albeit weakly, only when the DNA was relaxed with topoisomerase subsequent to UV endonuclease III treatment.

Attempt to Identify Lesions Recognized in Heavily Irradiated DNA

Certainly, UV endonuclease III recognizes baseless sights formed by heavy UV irradiation, but it also appears to act at some alkali-stable sites in this DNA. We have attempted without success to characterize these lesions. Both the murine and the human fibroblast UV endonuclease III act preferentially on supercoiled, UV-irradiated DNA. When such DNA is relaxed with topoisomerase, the rate of nicking of the substrate was 22 and 35% optimal for the murine and human enzymes, respectively. Moreover, we rarely detected more than two or three nicks per molecule, regardless of the UV dose. This preference for closed, circular DNAs precludes easy identification of the lesion(s) recognized by the enzyme without having more specific substrates available. Using several such substrates, we have shown that the mammalian preparations have no detectable uracil-, 5-hydroxylmethyluracil, or FAPY DNA glycosylase activity. When very radioactive [thymidine-H]DNA was prepared by nick translation and then irradiated, some release of radioactivity was detected, but HPLC analysis of the label released did not identify conclusively the several peaks to be any known base damages, both because of limited material and because oxidized forms of thymine are not well resolved by HPLC. Given the very limited activity of this enzyme due to its preference for closed, circular DNA and its apparent preference for uncommon lesions, baseless sites remain the only characterized substrate brought about by the heavy irradiation.

Relationship between UV Endonuclease III and Ribosomal Protein S3

UV Endonuclease III and AP Endonuclease I Activities Are Associated with Ribosomal Protein S3 (rpS3)

UV endonuclease III from MPC-11 cells was purified to yield a single visible protein band of M = 32,000 on denaturing gels. During the purification, both UV and AP endonuclease activities copurified with this band. Three peptides obtained from this protein by treatment with V8 protease were purified and sequenced: KRFGFP, KVATRGLCAIAQAESL, and KGGKPEPPAMPQPV. These have 100% identity with sequences found in rat ribosomal protein S3 (Chan et al., 1990).

To confirm that nuclease activity was indeed associated with the ribosomal protein, affinity-purified antibody to rat ribosomal protein S3 and a cDNA clone for the rat protein were utilized. In the first instance, two antibody preparations were shown to be non-neutralizing for the UV endonuclease III activity but to be able to immunodeplete it (). In addition, these antibodies were able to bind the protein associated with the activity onto protein A-Sepharose beads from which it could be extracted (Fig. 5). Hence, the affinity-purified antibodies, which were made against rat rpS3 isolated from ribosomes, recognized both the protein and the nuclease activity of the murine UV endonuclease III preparation.


Figure 5: Depletion with antibodies to ribosomal protein S3 of the protein associated with the nuclease activity. 20 µl of suspension of protein A-Sepharose CL (Pharmacia Biotech Inc.) suspended in 5% glycerol, 50 mM KCl, 25 mM Tris-HCl, pH 8.0, and 0.005% Triton X-100 were mixed with antibody as indicated and incubated for 90 min at 4 °C. The beads were then washed three times by centrifugation with 2-fold concentrated suspension buffer, and finally purified UV endonuclease III from MPC-11 cells was added and incubated with the beads for 1 h at 4 °C. The beads were then removed and washed by centrifugation. The initial supernatants (leftgel) and material bound by the beads (rightgel) were then probed by immunoblotting with antibody directed against rpS3. The supernatants were also assayed for enzyme activity (Table V). The protein that had bound to the protein A-Sepharose was released for the immunoblotting by boiling in the SDS buffer used for loading sample onto the SDS-polyacrylamide gel electrophoresis gel. Blots were developed with an alkaline phosphatase immunoblot kit (Bio-Rad). Tracks are M, unprocessed UV endonuclease III run on the gel; C, no enzyme added to the beads; Ab1 is antibody 71/83; Ab2 is antibody 67/67; CS is a control antibody, which had been directed against rat ribosomal protein S26.



In the second approach to confirm the association of the activities with rpS3, the cloned cDNA for rat ribosomal protein S3, which was isolated by Chan et al.(1990), was sub-cloned into the phage T7 expression vector, pT7-7 (Tabor and Richardson, 1985) to obtain pT7-7S3 (see ``Experimental Procedures''). Expression of the protein corresponding to the cloned cDNA was then induced in E. coli. The induced protein had the expected apparent molecular weight of 32,000 and was purified by chromatography upon DEAE-cellulose, S-Sepharose, and fast protein liquid chromatography Superose 12. The purified peptide cross-reacted with the antibodies to rpS3 as expected. It also had UV endonuclease activity (Fig. 6). Preparations of the rat rpS3 expressed in E. coli consistently have had UV and AP endonuclease activities in approximately a 1:1 proportion, the same ratio as found for UV endonuclease III isolated from mammalian cells.


Figure 6: Overexpression of rat ribosomal protein S3 in E. coli and assay for nuclease activity. Rat ribosomal protein S3 was overexpressed in E. coli, purified, assayed for UV endonuclease activity (toppanel), and displayed on a denaturing gel stained with silver (bottompanel) as described under ``Experimental Procedures.'' The profile shown is that of the final purification step, Superose 12 fast protein liquid chromatography. TracksM and L contain protein size markers.



The overexpressed rpS3 preparation purified from E. coli produced nicks on depurinated DNA and heavily irradiated DNA linearly with time, and these reactions were unaffected by the presence of MgCl or EDTA (data not shown). Only two endonuclease activities that are able to act on depurinated DNA and UV-irradiated DNA in EDTA are known to exist in E. coli: Fpg protein (FAPY DNA-glycosylase) and endonuclease III (Linn and Deutscher 1993). FAPY DNA-glycosylase activity was assayed for and was not present in the preparation of rat rpS3 expressed in E. coli. To rule out contamination by E. coli endonuclease III, immunoblotting of the purified, overexpressed rpS3 preparation with antibody made against E. coli endonuclease III was performed. No E. coli endonuclease III was detected when the recombinant rpS3 preparation was probed with E. coli endonuclease III antibody, although sufficient endonuclease activity was probed so that if E. coli endonuclease III were responsible for the activity in the preparation, it would easily have been detected. Since the presence was ruled out of the only two known E. coli nucleases that could have been responsible for the nuclease activity in the rat rpS3 preparation obtained from E. coli, we conclude that the nuclease activities on heavily UV-irradiated DNA and depurinated DNA are associated with the overexpressed rat ribosomal protein S3 itself (parenthetically, the presence of AP and UV endonuclease activity in the overexpressed rpS3 preparation also supports the conclusion that those same two activities found in the preparations from animal cells also are associated with the ribosomal protein rather than with a contaminant from the animal cells).

Perhaps most significant, however, was the fact that the rat rpS3 expressed in E. coli was able also to catalyze the apparent nicking of the phosphodiester bond within cyclobutane pyrimidine dimers. The protein expressed in E. coli produced little apparent nicking in lightly irradiated DNA unless the T4 UV endonuclease (ser) was subsequently added (, Experiment II). In this case, a number of nicks was again observed that equaled the number of cyclobutane pyrimidine dimers expected to have been present (, Experiment II). Since this unique type of activity observed with UV endonuclease III from MPC-11 cells (but neither reported to be present in E. coli nor observed by us in extracts from E. coli not containing the rpS3 plasmid() ) is present in the purified rat rpS3 expressed in E. coli, we conclude that all of the activities observed for mammalian UV endonuclease III are physically associated with ribosomal protein S3.

In summary, the results obtained with the antibodies taken together with those using the rpS3 cDNA clone strongly indicate that ribosomal protein S3 is indistinguishable from UV endonuclease III in endonuclease specificity, antigenicity, and primary structure. They are also consistent with the recent report of Wilson et al. (1994) that ribosomal protein S3 from Drosophila also has AP lyase activity.

Ribosomal Protein S3 Appears to be Located Both in the Nucleus and in the Cytoplasm

In situ Confocal immunolocalization using rpS3 antibodies revealed that rpS3 is present both in the cytoplasm and the nucleus but not in the nucleolus (data not shown). These results are in agreement with earlier studies on ribosome assembly, which showed that rpS3 joins to preribosomal particles after leaving the nucleolus but before transport to the cytoplasm (Hadjiolov, 1985). After ribosomes are fully assembled in the cytoplasm, rpS3 is apparently loosely bound to the 40 S subunit (Lutsch et al. 1990; Bommer et al., 1991) and exchanges freely among cytoplasmic ribosomes (Hadjiolov, 1985). The apparent facility with which rpS3 detaches from the ribosome in vivo is lost upon purification of ribosomes from cell extracts, however. We find that with isolated ribosomes, rpS3 can only be released from ribosomes under denaturing conditions, suggesting that some active process might operate in vivo to facilitate release of the protein. All of these data strongly indicate that rpS3 is normally present in the nucleus.

If rpS3 were to interact with DNA, it might at some time be recruited back into the nucleus from the cytoplasm. Two signals are possibly involved in the transport of rpS3 to the nucleus. Both the peptide and the nuclease activity associated with UV endonuclease III are strongly bound by concanavalin A affinity columns (data not shown). Lectin blots confirmed that the protein interacts with concanavalin A, as well as with Ulex europeus agglutinin and soy bean agglutinin; however, it does not interact with wheat germ agglutinin, Dolichos biflorus agglutinin, or ricin (data not shown). This lectin binding pattern suggests a nuclear or nuclear membrane localization (Hart et al., 1989).

Rat ribosomal protein S3 also contains a potential nuclear localization signal motif (Silver, 1991), KKRK, near the N terminus. Sequence comparison among the rpS3 proteins from various species reveals that this sequence is conserved among the eukaryotic rpS3 proteins but is absent from the prokaryotic homologs (Fig. 7). Either this signal or the glycosylation modifications could be utilized to mark the protein for transport to the nucleus, and future study in this regard is warranted.


Figure 7: Peptide sequences of ribosomal proteins S3. Sequences were obtained from GenBank.




DISCUSSION

Kuhnlein et al.(1978) reported that AP endonuclease activity from cultured human fibroblasts was resolved into two peaks, AP endonucleases I and II, by phosphocellulose column chromatography and that the activity corresponding to AP endonuclease I was missing from extracts of XP group D cells. AP endonuclease I passed through phosphocellulose, whereas AP endonuclease II (the major AP endonuclease activity, which was not accompanied by UV endonuclease activity) was retained by the resin. Subsequent study revealed that AP endonuclease I cleaves on the 3`-side of the AP site by a -lyase mechanism to produce a 3`-sugar terminus, which is not an efficient primer for E. coli DNA polymerase I (this paper), whereas AP endonuclease II (now called APE (Demple et al., 1991) or HAP1 (Robson et al., 1992)) cleaves on the 5`-side of the AP site to produce 3`-hydroxyl nucleotide and deoxyribose 5-phosphate termini (Mosbaugh and Linn, 1980).

Kudrna et al.(1979) observed that depurinated SV40 DNA transfects XP-D cells considerably more efficiently than it does normal cells. Hence, there is a phenotypic correlation with the absence of the AP endonuclease. These results were unexpected since AP endonuclease activity was presumed to be involved in the repair of AP sites. It may be, therefore, that at least in mammalian cells, cleavage of DNA at AP sites by a -lyase activity is not a DNA repair event in the classical sense. Instead, an alternative, novel function for this ubiquitous enzyme activity, such as forestalling DNA repair, may be worthy of consideration.

The cleavage of the phosphodiester bond within a cyclobutane pyrimidine dimer that was observed for rpS3 also calls for a novel function. The activity could relax DNA distortions brought about by the dimer, possibly then affecting the facility to replicate past the dimer as suggested also by Galloway et al.(1994). Replication past pyrimidine cyclobutane dimers is common in vivo in mammalian, especially rodent, cells. Perhaps, for example, both the -lyase and the intradimer nicking activities serve to delay or modulate DNA repair in an overstressed damage situation so as to avoid excessive manipulation of the genome. These activities could in this way contribute to regulating DNA repair processes rather than to participate in them directly.

On storage or during purification, UV endonuclease III from mammalian cells or expressed in E. coli loses its preference for DNA damage, i.e. its activity upon undamaged DNA increases and approaches that upon undamaged DNA. It also behaves as though it becomes less anionic during ion-exchange chromatography. Tycowski et al.(1993) reported that human U15A RNA is coded for within intron I of the rpS3 gene. We tested the possibility that U15A RNA (prepared by in vitro transcription) could re-establish the specificity for UV-irradiated DNA. Approximately half the nonspecific activity could be suppressed by addition of the U15A RNA, suggesting that some RNA-like species, but probably not U15A RNA, could be a specificity factor that suppresses activity upon undamaged DNA. Thus, while the gene for U15A RNA is within the rpS3 gene, there is no evidence for a functional interaction between the respective gene products.

Ribosomal protein S3 forms part of the domain on the ribosome where the initiation of translation occurs; it can be cross-linked to eukaryotic initiation factors eIF-2 (Westermann et al., 1979) and eIF-3 (Tolan et al., 1983), and it appears to be directly involved in ribosome-mRNA-aminoacyl tRNA interactions during translation (Bommer et al., 1991). However, rpS3 has also been proposed to have functions other than protein synthesis by several laboratories based upon observations made in those laboratories. Mutants in the gene encoding rpS3 in Drosophila have a recessive lethal and a dominant Minute phenotype, which suggested a possible other role (Andersson et al., 1994). As noted above, the small nucleolar RNA, U15A, is processed from an intron of the human gene, which suggested a relation between regulation of translation and RNA splicing (Tycowski et al., 1993). Finally, rpS3 is overexpressed in colorectal cancer cells, which suggested a role in cell immortality (Pogue-Geile et al., 1991). The observations in this paper now present a fourth such report.

RpS3 could be utilized for DNA repair while in the nucleus prior to assembly into ribosomes, or it might be recruited from functional ribosomes. However, it finds its way to the genome, we would expect rpS3 to be involved in DNA damage processing in some way by virtue of (1) the preferences of the nuclease activity for DNA damage and (2) by the recent observation that transfection of normal or Fanconi's anemia human cell lines with a rpS3 cDNA construct makes these cells considerably more resistant to the DNA cross-linking agents, mitomycin C and diepoxybutane.()

DNA repair processes appear at least in part to be coupled to transcription, presumably for monitoring of damage and possibly for checkpoint control and stress responses (Drapkin et al., 1994). Whether an involvement of rpS3 in DNA damage processing would reflect a coupling of translation to DNA repair is an intriguing possibility. SSL1, for example, has been implicated in both of these functions in yeast (Yoon et al., 1992). Finally, it is interesting to note that in E. coli, rpS3 is a DNA binding protein, the H protein, that appears to be associated with the nucleoid and is required for activity of several DNA metabolic enzymes in vitro (Bruckner and Cox, 1989).

The ERCC2 gene corrects the phenotype of XP-D cells after gene transfer (Flejter et al., 1992). ERCC2 shares 52% amino acid sequence identity with yeast RAD3, and both genes encode a protein with DNA helicase and ATPase activity (Weber et al., 1990; Sung et al., 1993). Why then don't we detect the rpS3 nuclease activities in any XP-D cells? The rpS3 cDNA sequence is unchanged in XP-D cells, and rpS3 is clearly present on ribosomes in XP-D cells (GenBank Accession Nos. U14990, U14991, U14992(1994)). Possibly, a subset of rpS3 molecules is differently subjected to post-translational modification in XP-D cells, or possibly, rpS3 is complexed differently to other macromolecules in XP-D cells, thus changing its chromatographic or catalytic properties. Both of these possibilities are currently being investigated.

  
Table: Comparison of UV endonuclease activity in phosphocellulose flow-through fractions from normal and XP-D cells

Fibroblasts were harvested from T-150 flasks, 40 for the normal cells and 15 for the XP-D cells. 10 mg of crude extract protein was processed for the lymphoblasts. Processing of extracts and assay procedures are described under ``Experimental Procedures.'' UV endonuclease III is taken as the activity passing through phosphocellulose, whereas UV endonucleases I plus II are the activities that bind to the resin.


  
Table: Product of UV endonucleases acting upon AP sites in DNA in concert with a class II AP endonuclease

After removal of approximately 0.5 pmol (Experiment A) or 1 pmol (Experiment B) of uracil from 0.35 nmol of [P, uracil-H]poly(dA-dT) with uracil DNA glycosylase, the glycosylase was inactivated for 5 min at 70 °C. The DNA was then incubated with murine UV endonuclease III (20 units, 40 min), human UV endonuclease III (4.5 units, 120 min), or HeLa AP endonuclease II (200 units, 10 min). These enzymes were inactivated for 5 min at 70 °C before a second incubation with enzyme where indicated. Reaction mixtures were adjusted to 10 mM MgCl for AP endonuclease II. The specific activity of the [P]dUMP residues was 6700 cpm/pmol for Experiment I and 600 cpm/pmol for Experiment II. The sugar phosphate products were separated and quantitated as described under ``Experimental Procedures.''


  
Table: UV endonuclease III activity on UV-irradiated DNAs

Reaction mixtures contained 40 mM Tris (pH 8.0), 50 mM KCl, 0.01% Triton X-100, 5 mM 2-mercaptoethanol, 4 mM EDTA, 20 µM PM2 DNA (nucleotide residues), and 1.9 units of murine UV endonuclease III hydroxylapatite fraction. Incubation was for 15 min at 37 °C.


  
Table: Effect of alkali and T4 UV endonuclease (Ser) on cyclobutane pyrimidine dimers exposed to UV endonuclease III

In Experiment I, reactions (0.2 ml) contained 40 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM 2-mercaptoethanol, 0.01% Triton X-100, 3 mM EDTA, 20 µM PM2 DNA (nucleotide residues), UV-irradiated with 23 J/m, 4 units of UV endonuclease III, and sufficient T4 endonuclease to convert half of the pyrimidine dimers to alkali-labile sites. 20 µM PM2 DNA-nucleotide in 0.2 ml is equivalent to 212 fmol of DNA molecules, and 23 J/m produces roughly two cyclobutane pyrimidine dimers per molecule. Thus, there were roughly 420 such dimers in the reaction. After incubation for 15 min at 37 °C (1st incubation), the reaction mixture was heated at 70 °C for 5 min to inactivate the first enzyme; then, the reactions were incubated with the second enzyme (added as indicated) for 10 min at 37 °C (2nd incubation). Samples treated with alkali were mixed with 0.3 M potassium phosphate (pH 12.4 with KOH), incubated for 2 h at room temperature, and then neutralized with 1 M potassium phosphate (pH 4 with HCl). Nicks were monitored by nitrocellulose filter binding. In Experiment II, reactions (0.1 ml) contained the same buffer components, except that dithiothreitol replaced 2-mercaptoethanol. 10 µM PM2 DNA, which had been UV-irradiated with 23 J/m to yield roughly 110 cyclobutane pyrimidine dimers per reaction and 0.15 unit of rat ribosomal protein S3 expressed in E. coli were used. Both incubations were for 20 min with 1.9 units of the T4 enzyme added in the second incubation where indicated. ND, not done.


  
Table: Immunodepletion of endonuclease activity with antibody directed against ribosomal protein S3

Immunodepletion experiments were those described in Fig. 5. UV endonuclease activities in the supernatants were assayed after exposing UV endonuclease III to antibody, which had been attached to protein A-Sepharose beads and centrifuging to remove the beads.



FOOTNOTES

*
This research was supported in part by U. S. Dept. of Energy Grant 2ER61458 and National Institutes of Health Grant P30ES011896. 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.

§
Current address: Dept. of Biology, Korea University, Seoul 136-701, Korea.

Supported by a fellowship from Fundaão de Amparo A Pesquisa no Estado de Sao Paulo (Brazil). Current address: Dept. of Biochemistry, Federal University, Curitiba-PR, 81351-990, Brazil.

**
Current address: Dept. of Biology, Technion, Haifa 32000, Israel.

§§
Supported by the Association pour la Recherche contre le Cancer.

¶¶
To whom correspondence should be addressed. Tel.: 510-642-7583; Fax: 510-643-5035.

The abbreviations used are: XP, xeroderma pigmentosum; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; rpS3, ribosomal protein S3.

J. Kim and S. Linn, unpublished results.

L. Chubatsu and S. Linn, unpublished results.

L. Herzing, C. Allen, and M. S. Meyn, unpublished results.


ACKNOWLEDGEMENTS

For supplying materials, we are indebted to Dr. Jacques Laval (FAPY-DNA glycosylase and its substrate), Dr. Joan Steitz (pS3 (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) ), Dr. Ira Wool (pGEM2S315), and Drs. Stephen Lloyd, Jon K. de Riel, and K. Valerie for clones of T4 UV endonuclease V (Ser). We thank Dr. M. S. Meyn (Yale University) for making his unpublished transfection data available to us, Dr. D. G. Burbee (General Atomic, San Diego) for making the unpublished yeast rpS3 sequence available to us, B. L. Raether for providing unpublished human rpS3 sequences, and Ann Fisher for expert tissue culture services.


REFERENCES
  1. Amacher, D. E., Elliott, J. A., and Lieberman, M. W.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1553-1557 [Abstract]
  2. Anderson, C. T., and Friedberg, E. C.(1980) Nucleic Acids Res. 8, 875-888 [Abstract]
  3. Andersson, S., Saeb-Larssen, S., Lambertsson, A., Merriam, J., and Jacobs-Lorena, M.(1994) Genetics 137, 513-520 [Abstract/Free Full Text]
  4. Andrews, A. D., Barrett, S. F., and Robbins, J. H.(1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1984-1988 [Abstract]
  5. Bommer, U. A., Lutsch, G., Stahl, J., and Bielka, H.(1991) Biochimie (Paris) 73, 1007-1019 [CrossRef][Medline] [Order article via Infotrieve]
  6. Boyce, R. P., and Howard-Flanders, P.(1964) Proc. Natl. Acad. Sci. U. S. A. 51, 293-300 [Medline] [Order article via Infotrieve]
  7. Bruckner, R. C., and Cox, M. M.(1989) Nucleic Acids Res. 17, 3145-3161 [Abstract]
  8. Chan, Y. L., Devi, K. R., Olvera, J., and Wool, I. G.(1990) Arch. Biochem. Biophys. 283, 546-550 [Medline] [Order article via Infotrieve]
  9. Cleaver, J. E.(1968) Nature 218, 652-656 [Medline] [Order article via Infotrieve]
  10. Demple, B., and Linn, S.(1982) Nucleic Acids Res. 10, 3781-3789 [Abstract]
  11. Demple, B., Herman, T., and Chen, D. S.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11450-11454 [Abstract]
  12. de Weerd-Kastelein, E. A., Keijzer, W., and Bootsma, D.(1972) Nat. New Biol. 238, 80-83 [Medline] [Order article via Infotrieve]
  13. Drapkin, R., Sancar, A., and Reinberg, D.(1994) Cell 77, 9-12 [Medline] [Order article via Infotrieve]
  14. Flejter, W. L., McDaniel, L. D., Johns, D., Friedberg, E. C., and Schultz, R. A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 261-265 [Abstract]
  15. Friedberg, E. C., and King, J. J.(1971) J. Bacteriol. 106, 500-507 [Medline] [Order article via Infotrieve]
  16. Galloway, A. M., Liuzzi, M., and Paterson, M. C.(1994) J. Biol. Chem. 269, 974-980 [Abstract/Free Full Text]
  17. Gates, F. T., and Linn, S.(1977) J. Biol. Chem. 252, 1647-1653 [Abstract]
  18. Hadjiolov, A. A.(1985) Cell Biol. Monogr., Vol. 12, Springer Press, Vienna
  19. Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G.(1989) Annu. Rev. Biochem. 58, 841-874 [CrossRef][Medline] [Order article via Infotrieve]
  20. Hoeijmakers, J. H. J.(1993) Trends Genet. 9, 211-217 [CrossRef][Medline] [Order article via Infotrieve]
  21. Kane, C., and Linn, S.(1981) J. Biol. Chem. 256, 3405-3414 [Abstract/Free Full Text]
  22. Kim, J., and Linn, S.(1988) Nucleic Acids Res. 16, 1135-1141 [Abstract]
  23. Kim, J., and Linn, S.(1989) J. Biol. Chem. 264, 2739-2745 [Abstract/Free Full Text]
  24. Kraemer, K. H., Andrews, A. D., Barrett, S. F., and Robbins, J. H. (1976) Biochim. Biophys. Acta 442, 147-153 [Medline] [Order article via Infotrieve]
  25. Kraemer, K. H., Lee, M. M., and Scotto, J.(1984) Carcinogenesis 5, 511-514 [Abstract]
  26. Kudrna, R. D., Smith, J., Linn, S., and Penhoet, E. E.(1979) Mutat. Res. 62, 173-181 [Medline] [Order article via Infotrieve]
  27. Kuhnlein, U., Penhoet, E. E., and Linn, S.(1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1169-1173 [Abstract]
  28. Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S.(1978) Nucleic Acids Res. 5, 951-960 [Abstract]
  29. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Paterson, M. C., Lohman, P. H., de Weerd-Kastelein, E. A., and Bootsma, D.(1975) Proc. Natl. Acad. Sci. U. S. A. 72, 219-223 [Abstract]
  30. Linn, S., and Deutscher, M. P.(1993) in Nucleases (Linn, S., Lloyd, R. S., and Roberts, R. J., eds) 2nd Ed., pp. 455-468, Cold Spring Harbor Press, Cold Spring Harbor, NY
  31. Loeber, G., and Kittler, L.(1977) Photochem. Photobiol. 25, 215-233 [Medline] [Order article via Infotrieve]
  32. Lutsch, G., Stahl, J., Kärgel, H.-J., Noll, F., and Bielka, H. (1990) Eur. J. Cell Biol. 51, 140-150 [Medline] [Order article via Infotrieve]
  33. Melton, D. A., Krieg, P. D., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R.(1984) Nucleic Acids Res. 12, 7035-7056 [Abstract]
  34. Mosbaugh, D., and Linn, S.(1980) J. Biol. Chem. 255, 11743-11752 [Free Full Text]
  35. Nakabeppu, Y., Yamashita, K., and Sekiguchi, M.(1982) J. Biol. Chem. 257, 2556-2562 [Abstract/Free Full Text]
  36. Pogue-Geile, K., Geiser, J. R., Shu, M., Miller, C., Wool, I. G., Meisler, A. I., and Pipas, J. M. I.(1991) Mol. Cell. Biol. 11, 3842-3849 [Medline] [Order article via Infotrieve]
  37. Robson, D. N., Hochhauser, D., Craig, R., Rack, K., Buckle, V. J., and Hickson, I. D.(1992) Nucleic Acids Res. 20, 4417-4421 [Abstract]
  38. Silver, P. A.(1991) Cell 64, 489-497 [Medline] [Order article via Infotrieve]
  39. Snapka, R. M., and Linn, S.(1981) Biochemistry 20, 68-72 [Medline] [Order article via Infotrieve]
  40. Sung, P., Bailly, V., Weber, C., Thompson, L. H., Prakash, L., and Prakash, S.(1993) Nature 365, 852-855 [CrossRef][Medline] [Order article via Infotrieve]
  41. Tabor, S., and Richardson, C. C.(1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078 [Abstract]
  42. Tolan, D. R., Hershey, J. W., and Traut, R. T.(1983) Biochimie (Paris) 65, 427-436 [Medline] [Order article via Infotrieve]
  43. Tycowski, K. T., Shu, M. D., and Steitz, J. A.(1993) Genes & Dev. 7, 1176-1190
  44. Weber, C. A., Salazar, E. P., Stewart, S. A., and Thompson, L. H. (1990) EMBO J. 9, 1437-1447 [Abstract]
  45. Weinfeld, M., Gentner, N. E., Johnson, L. D., and Paterson, M. C. (1986) Biochemistry 25, 2656-2664 [Medline] [Order article via Infotrieve]
  46. Westermann, P., Heumann, W., Bommer, U. A., Bielka, H., Nygard, O., and Hultin, T.(1979) FEBS Lett. 97, 101-104 [CrossRef][Medline] [Order article via Infotrieve]
  47. Wilson, D. M., III, Deutsch, W. A., and Kelley, M. R.(1994) J. Biol. Chem. 269, 25359-25364 [Abstract/Free Full Text]
  48. Yoon, H., Miller, S. P., Pabich, E. K., and Donahue, T. F.(1992) Genes & Dev. 6, 2463-2477

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