A Sequence in the N-terminal Region of Human Uracil-DNA Glycosylase with Homology to XPA Interacts with the C-terminal Part of the 34-kDa Subunit of Replication Protein A*

(Received for publication, October 2, 1996)

Toril A. Nagelhus Dagger §, Terje Haug Dagger , Keshav K. Singh par , Kylie F. Keshav **, Frank Skorpen Dagger , Marit Otterlei Dagger , Sangeeta Bharati Dagger , Tore Lindmo §, Serge Benichou Dagger Dagger , Richard Benarous Dagger Dagger and Hans E. Krokan Dagger §§

From the Dagger  UNIGEN Center for Molecular Biology, The Medical Faculty, Norwegian University of Science and Technology, N-7005 Trondheim, Norway, par  Johns Hopkins Oncology Center, Radiobiology Laboratory, Baltimore, Maryland 21287-5001, the § Department of Physics, Norwegian University of Science and Technology, N-7005 Trondheim, Norway, Dagger Dagger  INSERM U.332, 75014 Paris, France, and ** Drexel University, Department of Bioscience and Biotechnology, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.


INTRODUCTION

Uracil-DNA glycosylase (UDG)1 is the first enzyme in base excision repair for removal of uracil from DNA and its main function is probably to remove mutagenic uracil residues resulting from deamination of cytosine in DNA (1). The subsequent steps in the base excision repair pathway include, as the minimal enzymatic requirement in vitro, an apurinic/apyrimidinic endonuclease, a deoxyribophosphodiesterase activity (which may be contributed by DNA polymerase beta ), DNA polymerase beta , and a DNA ligase (2). In analogy to the complexity of the nucleotide excision repair pathway, base excision repair is likely to be more complex in vivo. This is in fact supported by the finding of an alternative, short patch pathway, requiring proliferating cell nuclear antigen and DNA polymerase delta  (3, 4). A catalytically fully active form of human UDG has been expressed in Escherichia coli (5) and structure-function relationships determined by site-directed mutagenesis and x-ray crystallography (6). These studies identified this form of human UDG as a one domain structure with a positively charged DNA-binding groove. UDGs are relatively small monomeric enzymes that, at least in vitro, do not require cofactors. However, UDG is preferentially associated with replicating SV40 minichromosomes, indicating a possible interaction with components of the replication machinery (7). The gene encoding the major human UDG, UNG, is transcribed predominantly late in the G1-phase, resulting in a 2-3-fold increase in UDG activity early in the S-phase (8). The cell cycle regulation is consistent with the presence of several putative regulatory elements detected in the UNG gene (9), including a putative element for binding of replication protein A (RPA) (10) reported previously in DNA repair genes in yeast (11). RPA is a trimeric protein required for initiation of DNA replication (12, 13), in the initial steps of nucleotide excision repair in physical interaction with XPA (14), as well as in recombination repair (15). RPA interacts with XPA both via the p70 subunit (RPA1) and the p34 subunit (RPA2) (16). RPA from non-mammalian species substitutes poorly for human RPA during initiation of SV40 DNA replication (17, 18), and C-terminally deleted human RPA2 is only marginally active (19). RPA2 is phosphorylated within the replication initiation complex early in the S-phase (20) or following DNA damage caused by ultraviolet light (21) or ionizing radiation (22), but the role of phosphorylated RPA2 remains unclear. A human homolog of RPA2, called RPA4, has been identified (23), but its function is not known. The tumor suppressor p53 physically interacts with and inhibits the function of RPA, and this interaction may be important for regulating the onset of the S-phase (24).

In the present work, we demonstrate an interaction between human UDG from the UNG gene (UNG) and RPA using the two-hybrid system in yeast, enzyme-linked immunosorbent assay (ELISA), and a UDG activity assay. The results show that RPA2, but neither the homolog RPA4 nor RPA1, binds to UNG. This interaction is dependent on the N-terminal presequence in UNG. The presequence, which is not necessary for the catalytic activity of UNG, contains a region of 23 amino acids with strong homology to the conserved RPA2-binding region of XPA. These results indicate a possible function for RPA in base excision repair.


EXPERIMENTAL PROCEDURES

Two-hybrid Assay

Yeast reporter strain HF7c, used in the two-hybrid system, contains two Gal4-inducible reporter genes, HIS3 and LacZ (MATCHMAKER Two-hybrid system, Clontech Laboratories Inc.). Plasmid vectors, pGBT9 and pGADGH, encoding the Gal4 DNA-binding domain, Gal4-BD, (Gal4 residues 1-147) and Gal4-activating domain, Gal4-AD (Gal4 residues 768-881), respectively, were used to express hybrid proteins. pGBT9 and pGADGH also contain the yeast Trp1 and the Leu2 genes, respectively, as selectable markers. To screen for protein interactions in the two-hybrid system, we used pGBTUNGDelta 28, constructed by insertion of UNGDelta 28-cDNA, which lacks the 28 N-terminal amino acids of the open reading frame, into EcoRI/SalI-digested pGBT9, and a Jurkat cell cDNA library, constructed in fusion with Gal4-AD in pGAD1318. The yeast reporter strain HF7c was sequentially cotransformed with the UNGDelta 28 hybrid expression plasmid and the Jurkat cell cDNA library (100 µg) according to Bartel and Fields (25). Interactions with the UNGDelta 28 hybrid protein were assayed as described by Clontech Laboratories Inc. Positive clones were further tested for specificity by retransformation into HF7c either with pGBTUNGDelta 28 or with extraneous targets as yeast pGBTSNF1 or pGBT9. The SNF1-Gal4-BD/SNF4-Gal4-AD interaction in the two-hybrid system was used as positive control (26), and plasmid vectors without insert were used as negative control. The cDNA inserts from positive clones were sequenced using primer walking and TaqPRISMTM Ready Reaction DyeDeoxyTM terminator cycle sequencing kit on an Applied Biosystems model 373A DNA sequencing system (Applied Biosystems). The human RPA2 hybrid with the Gal4-AD in pGADGH (isolated from a HeLa cDNA library, Clontech Laboratories Inc.) was kindly supplied by K. Tanaka (27). In order to identify amino acids involved in the interaction between UNG and human RPA2 (p34), pGBTUNGDelta 75 and pGBTUNGDelta 84 were constructed the same way as pGBTUNGDelta 28. Sequencing of the vectors were performed in order to ensure in frame reading. Yeast cells were cotransformed with pGAD-RPA2 and pGBT-UNGDelta 28 or pGBT-UNGDelta 75, and necessary controls were performed. The filter assay was performed according to Clontech Laboratories Inc. pACTRPA1 was constructed by cloning the RPA1 NcoI-SalI fragment from pRPA70 into the NcoI-XhoI sites of pACT2 (28). pACTRPA2 was constructed by cloning the RPA2 XhoI-EcoRI fragment from pJGRPA2 into the XhoI-EcoRI sites of pACT2. pACTRPA4 was constructed by cloning a PCR-generated fragment encoding the open reading frame of RPA4 into the BamHI site of pACT11. UNGDelta 28 was fused to the Gal4 DNA-binding domain in the pAS vector and called pASIUNGDelta 28. The beta -galactosidase assay was performed as described by Harper et al. (28).

Generation of [3H]Uracil Containing Substrate DNA Free of Single-stranded Gaps or Nicks

A 1-kilobase pair genomic fragment from the uracil-DNA glycosylase gene was amplified by PCR in the presence of [3H]dUTP (15 Ci/mmol) (Amersham Corp., Little Chalfont, Buckinghamshire, United Kingdom) instead of dTTP. PCR products were purified by gel filtration followed by adsorption to glass milk and subsequent elution according to the Geneclean II protocol (BIO 101, Inc., Vista, CA) in order to remove excess primers and free nucleotides. [3H]Uracil-containing DNA (60,000 dpm/µl and ~ 1 ng of DNA/µl) was stored in 1 × TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at -80 °C.

Purification of UNGDelta 28 and UNGDelta 84

Heterologous expression and purification of UNGDelta 84 (lacking 84 amino acids of the N-terminal part) have been described previously (5). UNGDelta 28 (lacking the N-terminal 28 amino acids in the presequence) was expressed using a Baculovirus system2 and purified to homogeneity as described for UNGDelta 84 (5). The UDG activity of UNGDelta 28 is severalfold lower than that of UNGDelta 84 on the substrate used in the present experiments.2 Purified proteins were stored in 50 mM Hepes, pH 8.0, 1 mM dithiothreitol, 0.125 M NaCl, and 50% glycerol at -20 °C.

Assays for the Effect of RPA on UDG Activity on Double- and Single-stranded DNA Substrate

To test the effect of trimeric RPA on UDG activity, UNGDelta 28 or UNGDelta 84 (10 ng) and recombinant trimeric RPA (200 ng), kindly provided by Dr. M. S. Wold (29), were incubated for 30 min on ice in 200 µl of 2 mM NaCl, 40 mM Tris acetate, pH 7.0, 1.6 mM MgCl2, 0.2 mg/ml bovine serum albumin, and 0.2 mM dithiothreitol. Then aliquots of the preincubation mixture were diluted in the same buffer to equal enzyme activities, mixed with double-stranded [3H]uracil-containing DNA prepared by PCR, and UDG activity measured as described (30).

To investigate whether RPA bound to single-stranded DNA could recruit UNG to DNA, 100 ng of RPA diluted in assay buffer was preincubated on ice for 30 min with rate-limiting amounts of heat-denatured [3H]uracil-containing single-stranded DNA prepared by nick translation of calf thymus DNA (30). This was done by diluting the assay volume to 200 µl such that the final concentration of [3H]uracil (in DNA) was approximately 0.27 µM, which is severalfold below Km for both forms of UNG. Then 40 pg of UNGDelta 84 or 3.5 ng of UNGDelta 28 were added, and the assay was mixture incubated at 37 °C for time periods specified in the results. UDG activity was determined as described in Ref. 30. To investigate whether RPA in solution could recruit UNG to DNA, UNG proteins and RPA were preincubated and then substrate was added under otherwise similar conditions.

Sandwich ELISA for UNG Binding to RPA

Wells of immunoplates (Nunc Maxisorb, Roskilde, Denmark) were coated with 1 µg of RPA in 100 µl of phosphate-buffered saline (PBS), pH 7.4, at 4 °C overnight. All subsequent incubations were done at room temperature. The wells were blocked by incubation with 1 mg of bovine serum albumin in 200 µl of PBS for 1 h and washed with PBS (three times with 200 µl/well). Then varying concentrations of UNGDelta 28 or UNGDelta 84 in 100 µl of PBS with 2% fetal calf serum were added to the wells, and the plates incubated for 1 h. After washing as above, protein A-purified rabbit antibodies raised against UNGDelta 84 (5) were added (15 ng/well). The anti-UNG antibodies (PU101) detect UNGDelta 28 and UNGDelta 84 equally well in an ELISA system when they are coated directly onto immunoplates.2 After further incubation for 1 h and subsequent washing with PBS, horseradish peroxidase-conjugated swine anti-rabbit IgG (Dacopatts A/S, Glostrup, Denmark) was added and binding quantitated using a colorimetric method (o-phenylenediamine, Dacopatts A/S) according to the manufacturer's instructions.

Digestion of UNG with Proteinase K

UNG proteins were treated with proteinase K, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted, and visualized as follows: 3.2 pmol of UNGDelta 28 (calculated molecular mass 30.9 kDa, SDS-PAGE molecular mass 33 kDA) or UNGDelta 84 (calculated molecular mass 25.5 kDa, SDS-PAGE molecular mass 27 kDa) diluted in UDG buffer (10 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin) were incubated with 400 ng of proteinase K (Sigma) in a total volume of 100 µl for 0 and 160 min at room temperature. At 160 min 100 µl of UDG buffer or UDG buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma) were added to the tubes which were immediately placed on ice. To test whether RPA protected UNGDelta 28 against protease digestion, 3.2 pmol of UNGDelta 28 diluted in UDG buffer were preincubated on ice with 8.6 pmol (1 µg) RPA in a total volume of 100 µl for 30 min. Then the sample was split in two equal parts, incubated with 400 ng of proteinase K or buffer for 160 min at room temperature, and digested UNG proteins analyzed by Western blotting using PU101 antibodies for detection (5).


RESULTS

Interaction between UNG and RPA2 (p34) in the Two-hybrid System

Using the hybrid protein Gal4-BD-UNGDelta 28, a human Jurkat cDNA library fused to Gal4-AD in pGAD1318 (gift from J. Camonis, U248, INSERM, Paris) was screened in the yeast two-hybrid system with a transformation efficiency of 2.5 × 106 transformants/100 µg of library DNA used. From a 5.6-fold amplification, 62 positive clones were identified. All these clones indicated an interaction between UNGDelta 28 and a protein closely related to RPA2 as determined by nucleotide sequencing. Initially we assumed that this was a new homolog of RPA2. However, this potential RPA2 homolog hybridized strongly to genomic DNA from rat and not to human DNA. Furthermore, the nucleotide sequence of this cDNA clone was identical to a partial cDNA clone for rat RPA2 given the accession number H32647[GenBank] in GenBankTM. Therefore this new RPA2 cDNA clone most probably results from contamination of the human cDNA library with cDNA from rat and has consequently been reported to GenBankTM (accession number X98490[GenBank]) as a putative rat homolog of human RPA2 showing 89% identity with the human protein. Using this information, interaction between the human hybrid proteins Gal4-BD-UNGDelta 28 and Gal4-AD-RPA2 (authentic human RPA2) was demonstrated with both His3 and LacZ reporter genes (Fig. 1, panel A). No interaction was detected between the Gal4-AD-RPA2 and Gal4-BD-UNGDelta 75 (Fig. 1, panel B) or Gal4-BD-UNGDelta 84 hybrid proteins (data not shown), indicating that the binding of RPA2 to UNG is dependent on the presequence of UNG. This interaction between RPA2 and UNG is specific, since the RPA2 hybrid did not interact with other irrelevant Gal4-BD hybrid proteins such as Gal4-BD-SNF1 or the unfused Gal4BD (Fig. 1A). In addition to the controls specified in Fig. 1, panels A and B, we tested for interaction between Gal4-BD-UNGDelta 28 and Gal4-AD or Gal4-AD-SNF4, but no such unspecific binding was observed (data not shown).


Fig. 1. Specific interaction of UNG with human RPA in the two-hybrid system. The reporter strain HF7c transformed with pGADp34 and pGBTUNGDelta 28 (panel A) or pGBTUNGDelta 75 (panel B) was analyzed for histidine auxotrophy and beta -galactosidase activity. Transformants were plated on medium with histidine, but without tryptophan and leucine (His+), replica-plated on medium without tryptophan, leucine, and histidine (His-), and on Whatman 40 filter on His+ medium in order to test for beta -galactosidase activity. Growth in the absence of histidine and blue colonies in the beta -galactosidase test indicate interaction between hybrid proteins. Interaction between SNF1 and SNF4 yeast proteins was used as a positive control. The interaction between UNGDelta 28 and RPA2 was also verified in a modified two-hybrid system (28), while no interaction between UNGDelta 28 and RPA1 or RPA4 was detected (panel C).
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Although no other interactions were observed after screening with the cDNA library, we investigated the possible interaction of UNGDelta 28 with RPA1 (known to interact with XPA) and RPA4 using a modified version of the two-hybrid system (28). Human RPA1, RPA4, and RPA2 (as a positive control) were each fused to the Gal4-AD in the pACT vector, and UNGDelta 28 was fused to the Gal4-BD in the pAS vector. As shown in Fig. 1C, UNGDelta 28 interacted only with RPA2 and not with RPA1 or RPA4. We did not directly test for possible interaction with RPA3, but such an interaction may not be likely since it could not be demonstrated when human UNG was screened against the Jurkat T-cell cDNA library.

The C-terminal Region of RPA2 Interacts with UNG

The longest and shortest RPA2-cDNA clones obtained from the screening, started at positions corresponding to amino acids 4 and 136 in human RPA2, indicating that the C-terminal region of RPA2 from amino acid residue 136-271 is sufficient for binding to UNG. UNG probably interacts with an amino acid region of RPA2 that is divergent in RPA4. Although RPA4 has a 47% overall identity with RPA2, a region located between positions 184 and 210 in RPA2 is distinctly different in the corresponding region in RPA4. Alignment of human RPA2 with RPA2 from other species shows that this region appears to be conserved in mammals, but not in lower eukaryotes or the human RPA2 homolog RPA4 (Fig. 2).


Fig. 2. Alignment of the possible UNG-binding region (position 184-210) at the C-terminal part of human RPA2 (accession number J05249[GenBank]) (first line) and corresponding regions in RPA2 from rat (accession number X98490[GenBank]) (second line), mouse (accession number D00812[GenBank]) (third line), S. cerevisiae (P22138[GenBank]) (fourth line), Crithidia fasciculata (accession number Z23164[GenBank]) (fifth line), and human RPA4 (accession number U24186[GenBank]) (sixth line).
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Binding of Trimeric RPA to UNG in Vitro and Effect on UDG Activity of UNG Proteins

To examine whether physiologically active trimeric form of RPA interacted with UNG in vitro, ELISA assays using UNG-Ab and purified recombinant trimeric RPA together with purified UNGDelta 28 or UNGDelta 84 were performed. Data shown in Table I support the UNGDelta 28-RPA interaction, while the severalfold lower absorbance detected with UNGDelta 84 may represent unspecific interaction close to the detection limit or very weak specific interactions. This experiment demonstrated that the RPA2-UNG interaction is direct and does not require a yeast intermediate protein. Furthermore, UNG binds to RPA2 in its heterotrimeric physiological context.

Table I.

Binding of UNG to trimeric RPA detected by ELISA

Wells of microtiter plates were coated with RPA, exposed to UNGDelta 28 or UNGDelta 84, and developed as described under "Experimental Procedures."
UNG added Binding of UNGDelta 28 (A492) Binding of UNGDelta 84 (A492)

pmol
8 0.290  ± 0.021 0.039  ± 0.012
0.8 0.042  ± 0.002 0.010  ± 0.002
0 0 0

We also tested the effect of trimeric RPA on UDG activity of UNG proteins on double-stranded DNA substrate. Since RPA binds strongly to single-stranded DNA, a UDG substrate without detectable gaps or single-stranded regions (generated by PCR) was used for this purpose. Using this substrate in the presence of excess trimeric RPA, UDG activity of UNGDelta 28 was inhibited by approximately 25%, while the UDG activity of UNGDelta 84 remained unaffected (Table II). These in vitro assays support the view that the interaction between the two proteins is specific and not an artifact observed with the hybrid protein in the two-hybrid system. Furthermore, the lack of effect of RPA on UNGDelta 84, which does not contain the presequence, is consistent with the results of the two-hybrid experiments.

Table II.

Effect of RPA on UDG activity on double-stranded [3H]uracil-containing DNA

RPA was preincubated with UNGDelta 28 or UNGDelta 84 and then incubated with [3H]uracil-containing DNA as described under "Experimental Procedures."
UDG activity
Ratio RPA/UNG UNGDelta 28 UNGDelta 84

%
0 100 100
20 75  ± 7 100  ± 10
200 75  ± 4 100  ± 8

In order to investigate whether the strong DNA binding capacity of RPA could mediate recruitment of UNG to single-stranded DNA, UDG assays were performed under conditions where the concentration of single-stranded DNA substrate was limiting. Neither preincubation of trimeric RPA with substrate nor preincubation of trimeric RPA with UNGDelta 28 or UNGDelta 84 resulted in consistently increased UDG activity. Rather, a weak inhibition of UDG activity was mostly observed under the varying conditions used; thus we could not demonstrate significant recruitment of UNG by RPA in these in vitro experiments (Table III).

Table III.

Effect of RPA on UDG activity of UNG proteins on single-stranded [3H]uracil-containing DNA present in limiting amounts ("recruitment experiment")

RPA was either preincubated with UNGDelta 28 or UNGDelta 84 prior to addition of substrate (RPA/UNG), or preincubated with substrate prior to addition of UNGDelta 28 or UNGDelta 84 (RPA/DNA). UDG activity in the absence of RPA was set to 100%.
Time RPA UNGDelta 84
UNGDelta 28
RPA/UNG RPA/DNA RPA/UNG RPA/DNA

min activity in % activity in % 
10  - 100  ± 8 100  ± 9 100  ± 7 100  ± 4
10 + 87  ± 5 114  ± 5 89  ± 6 85  ± 6
20  - 100  ± 3 100  ± 1 100  ± 7 100  ± 10
20 + 83  ± 1 127  ± 12 93  ± 4 80  ± 8

The RPA2-binding Region in UNG Is Homologous to the RPA2-binding Region in XPA

The region of XPA required for interaction with RPA2 is located within the N-terminal 58 amino acid residues (16). Interestingly, within the 49 amino acids of the presequence of UNG, which interacts with RPA2, a 23-amino acid region with strong homology to the RPA2-binding region of XPA is found. The hydrophobicity/hydrophilicity profiles are also very similar. When allowing for two gaps of 2 amino acids, 11 out of 23 amino acids are identical (47.8% identity), and in addition 3 are conserved (60.9% similarity), (Fig. 3, panel A). XPA from several other species also show homology to this region of human UNG with chicken showing the highest homology (60.9% identity and 78.3% similarity). SV40 T-antigen, which also binds to the C-terminal region of RPA2 (19), has a 26-amino acid region with limited homology to human UNG. In addition, a protein in the cyclin family (UNG2) apparently having UDG activity also has a region of homology to UNG and XPA (Fig. 3B). The presequence of mouse UDG3 is also strongly homologous to human UDG and contains the putative RPA2-binding motif. The corresponding region is also conserved in UDG from the fish Xiphophorus, although more distantly related,4 but is absent in UDG from yeast or animal viruses (various herpes simplex viruses and pox viruses) (data not shown). Although limited to a small region, this is, to our knowledge, the first homology demonstrated between damage recognizing proteins in the nucleotide excision repair and base excision repair pathways.


Fig. 3. Panel A, alignment of the putative RPA2-binding regions in UNG and XPA. The hydrophobic/hydrophilic properties of the aligned regions are represented with Goldman, Engelman, and Steiz (GES) curves (44). Panel B, alignment of the putative RPA2-binding region in human UDG (UNG1, accession number P13051[GenBank]) (first line), human XPA (accession number P23025[GenBank]) (second line), chicken XPA (accession number P27089[GenBank]) (third line), Xenopus laevis XPA (accession number P27088[GenBank]) (fourth line), mouse XPA (accession number S41498[GenBank]) (fifth line), Drosophila melanogaster (accession number P28518[GenBank]) (sixth line), human UNG2 (accession number P22674[GenBank]) (seventh line), and SV40 large T-antigen (accession number P03070[GenBank]) (eighth line).
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In conclusion, these results support the biochemical data and suggest that residues 57-79 in human UNG and 20-46 in XPA interact with RPA2.

The RPA2-binding Presequence in UNG Probably Constitutes a Separate Structural Domain

The crystal structure of UNGDelta 84, a catalytically fully active enzyme, has revealed a globular one-domain protein with the N-terminal region localized on the surface. Using proteinase K digestion, the presequence is easily removed from UNGDelta 28, resulting in a processed form slightly longer than UNGDelta 84 (Fig. 4, lanes 2 and 3). No further digestion of UNGDelta 28 is observed. Purified UNGDelta 84 is proteinase K-resistant, probably due to its compact structure (lanes 2 and 4). In an attempt to examine whether binding of RPA to UNGDelta 28 affected the proteinase K digestion, UNGDelta 28 was preincubated with RPA and then incubated with proteinase K. Lanes 2 and 6 show that RPA did not protect UNGDelta 28 from protease digestion. Proteinase K treatment of UNGDelta 28 removed the presequence without any loss of activity, in fact the activity is increased (data not shown), whereas the catalytic activity of UNGDelta 84 was not affected by the proteinase K treatment. The shorter form of UNG resulting from proteinase K treatment could not have resulted from digestion at the C-terminal end, because deletion of only the last 4 amino acids at the C-terminal end by site-directed mutagenesis where Trp-301 was converted to a stop codon resulted in complete loss of activity (data not shown). These results, together with the results obtained in the two-hybrid studies, indicate that the presequence constitutes a separate structural domain required for RPA binding, but not for catalytic activity.


Fig. 4. Western blot analysis of proteinase K-digested UNG. Lane 1, Undigested UNGDelta 28; lane 2, UNGDelta 28 digested with proteinase K; lane 3, undigested UNGDelta 84; lane 4, UNGDelta 84 digested with proteinase K; lane 5, UNGDelta 28 preincubated with RPA only; lane 6, UNGDelta 28 preincubated with RPA and digested with proteinase K.
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DISCUSSION

In this report we demonstrate an interaction between human UNG and human RPA in vivo, using the yeast two-hybrid system, and in vitro by an ELISA assay using purified trimeric RPA and purified forms of human UDG (UNGDelta 28 or UNGDelta 84). Only the 34-kDa subunit of RPA2 interacts directly with UNG, and the interaction results in a weak inhibitory effect on UDG activity rather than stimulation. The weak inhibitory effect may be an in vitro effect, and we do not suggest that RPA inhibits UDG activity in the much more complex in vivo situation; this result merely strengthens the other results showing that the presequence is involved in an interaction between UNG and RPA, since a possible functional consequence on UDG activity should be confirmed inside cells. Previously, RPA has been shown to be involved in the damage recognition step of nucleotide excision repair (14), in recombination repair (15), and in replication (13), but a possible involvement in base excision repair has not been reported, except for a possible role of RPA as a transcription factor in the regulation of expression of DNA repair genes (11). A putative element for binding of RPA is located in an inhibitory region of the UNG promoter (9, 10). In nucleotide excision repair, XPA interacts with both RPA2 and RPA1, but whereas RPA1 is essential for cell survival after UV light exposure, RPA2 has a more modest although significant effect (16). A region between amino acids 28 and 78 in UNG is necessary for interaction with RPA2, but we have not formally shown that this region is sufficient for interaction. Interestingly, amino acids 57-79 in UNG show strong homology to amino acids 20-46 in human XPA. Since XPA residues 1-59 are necessary and 1-74 sufficient for binding to RPA2 (16), it seems likely that the region of homology between amino acids 57-79 in human UNG and amino acids 20-46 in XPA are the actual RPA2-binding regions. At the C-terminal end of the region of homology, the amino acid sequence RLAAR is present in both UNG and XPA. However, this sequence does not appear to be sufficient for RPA2 binding, since Gal4-BD-UNGDelta 75 that starts with this sequence did not interact in the two-hybrid system. Our results indicate that the interacting region in human RPA2 is located downstream of the first 136 amino acid residues. A conserved region in RPA2 from mammals, lacking in RPA4 and RPA2 from lower eukaryotes, is present between residues 184 and 210. This may be a candidate region for binding of UNG, and if so, the pattern of conservation would indicate that only RPA from higher eukaryotes will recognize UNG, analogous to the ability of RPA from mammals, and inability of RPA from lower eukaryotes like Saccharomyces cerevisiae to bind to T-antigen and to stimulate SV40 replication (17, 18).

One possible function of RPA2 could be to recruit UNG to a DNA repair complex possibly associated with the replication complex for scanning for uracil prior to replication. A related function has been proposed for SV40 T-antigen which, like dnaC, lambda  P protein, and the T4 phage gp59 protein, facilitate primase recognition of RPA-coated DNA (Ref. 18 and references therein). Alternatively, RPA2 might assist transport of UNG either to the nucleus or within the nucleus. Among the three subunits, only RPA2 is associated with the nuclear matrix and this association persists throughout the cell cycle (31). In addition, RPA2 and RPA1 undergo a transition from uniform nuclear distribution to the punctuate nuclear pattern typical for replication foci during the S-phase (32). Nuclear staining of UNG also displays a nonhomogeneous distribution reminiscent of replication foci (33), although it has not been shown that UNG and replication foci colocalize. One of the functions of RPA2 could be to contribute to the localization of UNG in replication foci. UDG activity is not essential for DNA replication in E. coli or S. cerevisiae (34, 35). In contrast, it is essential for poxvirus replication (36) and for the replication of a herpes virus (HSV1) in mammalian cells that do not express cellular UDG (37). In human cells, UNG is most actively transcribed very late in the G1-phase and UDG activity subsequently increases 2-3-fold early in the S-phase (8). In addition, an association of UDG activity with replicating SV40 minichromosomes (7) may indicate a possible function for UNG in replication or in an associated repair complex.

The protease digestion in the present study indicates that the first approximately 80 amino acids, comprising the presequence of UNG, constitute a second structural domain separated from the previously reported structure of the catalytic domain by at least one protease-accessible site (6). We have reported previously that the nuclear and mitochondrial form of human UDG have a size corresponding to the "mature" form lacking the presequence of 77 amino acids, while a larger form that reacted with an antibody specific for the presequence was present in the cytosol fraction. In addition, transfection experiments indicated that the presequence was essential for mitochondrial import, but not for nuclear import (38). However, there are some inconsistencies in the literature regarding the size of nuclear UDG, since some reports indicate a size consistent with processing of the N-teminal end (39, 40), whereas others report a size essentially corresponding to the larger "preform" (41, 42). In conclusion, we do not yet know whether processing at the N-terminal end is a physiological process or merely an in vitro artifact during purification. Proteolytic cleavage of UDG during purification has been reported in yeast (43), and a similar digestion of human nuclear UNG may take place. However, the interaction of the "presequence" of UNG with RPA, a truly pleiotropic nuclear protein, necessitates further studies on the biochemistry of nuclear uracil-DNA glycosylase and possible accessory proteins assisting in base excision repair.


FOOTNOTES

*   This work was supported by The Research Council of Norway, The Norwegian Cancer Society, and the Cancer Fund at the Regional Hospital, Trondheim.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X98490[GenBank] (rat RPA2).


   These authors contributed equally to this article.
§§   To whom correspondence should be addressed. Tel.: 47-73-598680; Fax: 47-73-598705.
1   The abbreviations used are: UDG, uracil-DNA glycosylase; RPA, replication protein A; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
2   S. Bharati, M. Otterlei, G. Slupphaug, and H. E. Krokan, unpublished results.
3   H. Nilsen, unpublished results.
4   R. B. Walter, personal communication.

Acknowledgments

We thank Dr. R. B. Walter and H. Nilsen for communicating unpublished results to us, Dr. K. Tanaka for supplying pGADGH, Dr. M. S. Wold for supplying purified trimeric human RPA, Dr. G. Slupphaug for supplying UNG-antibodies, and Dr. J. Camonis for supplying the Jurkat cDNA library.


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