©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Polymerase Conducts the Gap-filling Step in Uracil-initiated Base Excision Repair in a Bovine Testis Nuclear Extract (*)

(Received for publication, August 5, 1994; and in revised form, October 26, 1994)

Rakesh K. Singhal Rajendra Prasad Samuel H. Wilson (§)

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, , and could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.


INTRODUCTION

During the life span of any organism, genomic DNA can be damaged by various physical or chemical agents, and for faithful reproduction, all or most of these damaged DNA sites must be repaired. Organisms have complex systems for repairing DNA lesions including the following: direct lesion removal, recombination, base excision repair, methylation-directed mismatch repair, and nucleotide excision repair of bulky adducts(1, 2, 3, 4, 5) . In cases where DNA repair is carried out by the base excision repair (BER) (^1)pathway, a damaged or inappropriate base is excised from DNA and replaced by the base pair complementary nucleotide. DNA synthesis to replace the nucleotide involves incorporation of only one or a few dNMP residues (6, 7) . In mammalian systems, information on the role of any one of the five cellular DNA polymerases in DNA repair has been largely restricted to evidence obtained from inhibitor studies(8) . Since the DNA polymerase inhibitors, such as aphidicolin, dideoxynucleoside, N-ethylmaleimide, or antibodies, can inhibit more than one DNA polymerase, interpretations on involvement of a DNA polymerase in any one DNA repair mechanism have generally been confounded(8) . The present study was designed, first, to unequivocally determine whether there is a role for beta-pol in mammalian BER, second, to develop a system in which mammalian BER can be reconstituted from purified proteins, and third, to test the performance of other DNA polymerases in mammalian BER in vitro. Wiebauer and Jiricny (9) demonstrated involvement of beta-pol in a G:T-initiated base excision repair reaction by HeLa nuclear extract, using both anti-beta-pol antibody and dideoxynucleotide inhibition. Matsumoto and Bogenhagen (10) argued that beta-pol may be responsible for gap filling during repair of a tetrahydrofuran lesion by BER in a Xenopus laevis oocyte extract, and more recently Dianov et al.(11) , using a human cell nuclear extract system for uracil-initiated BER, obtained inhibition by dideoxynucleotide; this led these workers to conclude that beta-pol was responsible for the DNA synthesis step. Although these studies pointed to a role of beta-pol in short-patch DNA repair, more specific studies were required to settle the question of beta-pol involvement because of the following considerations. The polymerase requirement in the lymphoblastoid cell line-based BER system studied by Dianov et al.(11) was not clear cut. First, the reaction was inhibited by the alpha-pol inhibitor aphidicolin and only partially blocked by the beta-pol inhibitor ddNTP, yet purified mammalian beta-pol is not inhibited by 100 µg/ml aphidicolin and is >95% inhibited by ddNTP at a ddNTP/dNTP ratio of only 10(12, 13) . Further, it is known that alpha-pol is inhibited by ddNTP, at a high ratio of ddNTP to dNTP and can exhibit only partial inhibition by aphidicolin(13) . Thus, the inhibition pattern of the BER system studied by Dianov et al.(11) tends to confound the interpretation that beta-pol was involved. Second, the beta-pol requirement for the HeLa extract G:T-initiated BER system described by Wiebauer and Jiricny (9) was assigned with a beta-pol antibody that is non-neutralizing and relatively low titer. Although this antibody, under appropriate conditions, can specifically recognize beta-pol in a crude extract, use of a high titer, beta-pol-specific, neutralizing antibody would strengthen the conclusion that beta-pol is included in BER. Third, the picture concerning beta-pol involvement in BER was further complicated recently by the discovery of a beta-pol-like DNA polymerase in the yeast Saccharomyces cerevisiae along with findings by Wang et al.(14) . These workers developed a S. cerevisiae in vitro system for uracil-initiated BER and found that repair synthesis for osmium tetroxide and UV-damaged DNA was conducted by DNA polymerase . This finding is interesting in light of the presence of a beta-pol like enzyme in S. cerevisiae (DNA polymerase IV) and the observation that a deletion strain for the corresponding gene has no apparent phenotype(15, 16) . Hence, the studies with the S. cerevisiae system provided no indication of a beta-pol involvement in BER, and Wang et al.(14) have suggested that results on yeast DNA polymerase requirements in DNA repair may be extrapolated to mammalian DNA repair. In light of these ambiguities and apparent contradictions, and earlier demonstrations that purified beta-pol can completely fill short gaps in vitro(17, 18, 19) , we undertook a study to further examine the putative role of beta-pol in uracil-initiated base excision repair. To approach the question, we developed a crude nuclear extract-based in vitro BER system and two neutralizing polyclonal antibodies to beta-pol. We chose uracil-initiated repair as our model for BER because it is a well documented pathway in eukaryotic cells, and several of the mammalian enzymes likely to be involved are available as recombinant proteins, including uracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease, and DNA ligase I (for reviews, see (20, 21, 22) ).

Uracil arises in DNA by two independent pathways: first, deamination of cytosine to uracil occurs spontaneously or in response to oxidizing chemical agents such as sodium bisulfite (3) and nitric oxide(23) , giving rise to G:U mismatch. Second, higher levels of dUTP in the cell are associated with incorporation of dUMP into DNA opposite a template A. In the case of the G:U mismatch, there is mutagenic potential if the mismatch is not corrected, leading to the G:CA:T transition mutation. To establish an in vitro system, we used a synthetic oligonucleotide-containing uracil at a defined position, along with appropriate sites for restriction enzyme analysis of products. Our system is based on a bovine testis crude nuclear extract (24) . This in vitro system promotes robust base excision repair to convert the G:U mismatch to G:C. Our results indicate that DNA polymerase beta is solely responsible for the single nucleotide gap-filling synthesis in uracil-initiated BER in bovine testis nuclear extracts but not in S. cerevisiae.


EXPERIMENTAL PROCEDURES

Materials

Radiolabeled nucleotides were from DuPont NEN or ICN Radiochemicals. Electrophoresis grade acrylamide and bisacrylamide were from Bio-Rad. dNTPs, ATP, dideoxy-CTP (ddCTP), bovine serum albumin, T4 polynucleotide kinase, and restriction endonucleases were from Boehringer Mannheim. Formamide and urea were from Life Technologies, Inc. Human DNA polymerase alpha was from Molecular Biology Resources, Inc. Human placental PCNA and DNA polymerases and were a generous gifts from Dr. M. Lee, University of Miami, FL. Recombinant human and rat DNA polymerases beta were purified as reported earlier (25, 26) . M13mp18(+) ssDNA and the M13 universal sequencing primer were from Pharmacia Biotech Inc. Purified synthetic oligodeoxynucleotide primers complementary to various regions of the multicloning site of M13mp18 ssDNA (and synthetic template) were from Genosys Biotechnologies, Inc. S. cerevisiae strains were kindly provided by Dr. L. Prakash.

Buffers

Homogenization buffer A was 10 mM HEPES, pH 8.0, 1.5 mM MgCl(2), 10 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 10 mM sodium metabisulfite. Buffer B was 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 10 mM sodium metabisulfite, while buffer C is buffer B with 5% (v/v) glycerol.

Nuclear Extract Fractionation

Nuclear Extract

Frozen bovine testis was from J. Schmidt Co. (Baltimore, MD) or Pel-Freeze Biological (Rogers, AR) and held at -80 °C until use. Nuclear extract was prepared as reported earlier(24) . The final fraction, after 40% (NH(4))(2)SO(4) precipitation and dialysis against buffer B, is referred to as nuclear extract (Fraction I) and was stored at -80 °C until further use.

Mono Q and Mono S Ion-exchange Chromatography

Mono Q and Mono S ion-exchange columns (1 times 10 cm) were attached in tandem and equilibrated with buffer C. These columns were run at a constant flow rate of 10 ml/h using a LKB pump. Approximately 15 ml (approx100 mg of protein) of nuclear extract (Fraction I) was loaded on the column where it first passed through Mono Q and then over the Mono S ion-exchanger. The columns were washed with two to three column volumes of buffer C. The two columns were then separated, and the Mono S column was eluted with buffer C containing 1 M KCl. The fractions containing most of the eluted proteins were dialyzed overnight against 100 volumes of buffer C, centrifuged at 12,000 times g for 5 min to remove any insoluble material, and then concentrated with a Centricon-10 (Amicon) at 3,000 times g to the desired volume (Fraction II).

Gel Filtration Column Chromatography

Fraction II, 200 µl, was loaded on a LKB FPLC Superose 12 HR 10/30 column pre-equilibrated with Buffer C containing 1 M KCl, and the column was run at a flow rate of 0.25 ml/min. Fractions (0.5 ml) were concentrated with Centricon-10 concentration units and washed with buffer C to remove salt, and concentrated fractions were stored at -80 °C until further use. To standardize the column, 40 µl of Bio-Rad gel filtration molecular weight marker standard mixture containing thyroglobulin, bovine -globulin, chicken ovalbumin, equine myoglobin, and vitamin B-12 (670,000-1,350 kDa) was diluted to 200 µl in buffer C with 1 M KCl. The standard solution, 200 µl, was loaded on a FPLC Superose 12 column and run under similar condition, and 0.5-ml fractions were collected.

Base Excision Repair and Product Analysis

The reaction conditions for base excision repair by bovine testis nuclear extract were similar to those reported earlier(11) . Standard reaction mixtures (50 µl) contained 100 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 1 mM dithiothreitol, 0.1 mM EDTA, 2 mM ATP, 0.5 mM NAD, three deoxynucleoside 5`-triphosphates at 20 µM each, 5 mM diTris-phosphocreatine, 10 units of creatine phosphokinase, 40 nM of duplex oligonucleotide, 2-20 µM of the fourth [alpha-P]dNTP (specific activity approx10^5-10^6 disintegrations/min/pmol) and 50 µg (protein) of crude nuclear extract. Reactions were incubated for 10 min at 37 °C and stopped by addition of EDTA and NaCl to final concentrations of 50 mM and 0.3 M, respectively. The DNA was extracted with phenol-chloroform and precipitated with three volumes of non-chilled ethanol. The DNA precipitate was collected immediately by centrifugation to minimize co-precipitation of dNTPs, dried under vacuum, and resuspended in 10 µl of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. The duplex oligonucleotide (0.1-0.2 pmol) was incubated for 1 h at 37 °C with 20-50 units of restriction endonuclease under the conditions recommended by the manufacturer. Unless otherwise specified, reactions were carried out in the presence of 40 µCi of [alpha-P]dCTP (3000 Ci/mmol) alone, and the reaction was stopped by adding an equal volume of ``stopping solution'' (40 mM EDTA and 80% formamide) to the reaction mixture. After incubation at 95 °C for 2 min, the DNA was separated by electrophoresis on a 12% polyacrylamide gel containing 7 M urea in 89 mM Tris, 89 mM boric acid, and 2 mM EDTA (pH 8.8). Gels were fixed, dried, and autoradiographed to visualize the reaction products.

beta-Pol Assays

beta-Pol assays were conducted as described earlier(19) . M13mp18(+) ssDNA was used as template and M13 universal sequencing primer (17 nt) along with 51-mer synthetic primer were annealed to create a 5-nt gapped substrate.

Preparation of Antibodies

Antisera specific for intact rat beta-pol and its ssDNA binding 8 kDa domain (27) were raised by immunization of rabbits as described (28) . Recombinant rat beta-pol and 8 kDa domain were purified as reported previously(26) . Specific antibodies to beta-pol and 8 kDa domain were purified by affinity chromatography, using Epoxy-activated Sepharose 6-B gel (Pharmacia) coupled with beta-pol or 8 kDa domain. Coupling of the ligand and subsequent elution of the bound antibodies from the gel were conducted as suggested by the manufacturer. Antigenic and chemical characterizations were performed using appropriate dilutions of the affinity-purified antibodies to beta-pol or 8 kDa domain. These affinity-purified polyclonal antibodies to beta-pol and 8 kDa domain are referred to as anti-beta-pol and anti-8 kDa, respectively. The IgG fraction of the preimmune serum was purified by protein A-Sepharose Cl-6B (Pharmacia) column.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis

SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli(29) , using 9.5% discontinuous slab gels. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad) in a transblot apparatus (Bio-Rad), according to the manufacturer's instructions. Subsequently, the nitrocellular membrane was probed with affinity-purified anti-beta-pol (1:10,000) or preimmune IgG. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) at 1:10,000 dilution was used as the secondary antibody and was detected with an ECL chemiluminescence system (DuPont).

Uracil Base Excision Repair in S. cerevisiae

Nuclear extract was prepared from the S. cerevisiae wild type strain LP3041-6D (MAT alpha leu 2-3 leu 2-112 trp 1 Delta ura 3-52) and the isogenic beta-pol gene deletion strain JWY355 (MAT alpha leu 2-3 leu 2-112 trp Delta ura 3-52 beta-pol Delta::URA 3) exactly as described by Wang et al.(14) . The base excision repair reaction was carried out as with testis nuclear extracts.


RESULTS

Base Excision Repair with the Bovine Testis Nuclear Extract

To establish an in vitro system for base excision repair, we surveyed various sources of extract and substrate molecules. The bovine testis nuclear extract was selected because it has abundant uracil-initiated BER activity as will be described below and therefore will be an excellent starting fraction for purification of repair proteins. We used a synthetic oligonucleotide molecule designed so that we could readily study single nucleotide gap filling during repair. Incorporation of [alpha-P]dCMP during the BER reaction was carried out on a 51-residue duplex oligonucleotide substrate containing a U residue at position 22 in the lower strand (Fig. 1A). An autoradiogram illustrating the time course of the repair reaction is shown in Fig. 1B, panel A. Radiolabel corresponding to the 51-nucleotide product molecule appeared as a discrete band after 2 min of incubation, and an increase in product was seen with increasing time of incubation. At the longest interval, some product bands shorter than 51 residues appeared, which may have been the result of nuclease activity in the nuclear extract. We adjusted the concentration of DNA substrate and time of incubation so that the product formed corresponded to no more than 25% of the DNA substrate in the reaction mixture. We first examined the questions of whether this incorporation of [P]dCMP was specific to the G:U mismatch and if other dNTPs could substitute for dCTP. As shown in Fig. 1B, panel B, the repair reaction was carried out separately in the presence of different alpha-P-labeled dNTPs using either G:U or G:C containing duplex (51 bp) oligonucleotide substrates, differing only at position 22 in the lower strand. Incorporation of radiolabel into full-length product molecules occurred only with the substrate carrying the G:U mismatch, and [P]dCMP was the only nucleotide incorporated among those tested (Fig. 1B panel B).


Figure 1: A, sequences of duplex oligonucleotide substrates. Lower strand (LS) contained dUMP at position 22, relative to the 5` end G at position 1. Substrate containing dCMP in place of dUMP served as a reference. Restriction endonuclease sites, along with upper (US) and LS designations, are indicated. The last nucleotide (position 51 or 3` end) of the lower strand was ddATP in all substrates. Four other oligonucleotide substrates (not shown) were prepared using the identical sequence except that G:T, A:T, A:C, or A:U base pairs were created at position 22 where T, T, C, and U were in the lower strand, respectively (see Fig. 3). B, a composite figure showing uracil base excision repair assay with bovine testis nuclear extract. The repair reaction was carried out as described under ``Experimental Procedures.'' Autoradiograms of typical gel electrophoresis results are shown. PanelA, time course of product accumulation for the standard repair reaction. Aliquots were withdrawn at various times of incubation as indicated above each lane. Panel B, specificity of nucleotide incorporation in the repair reaction. The repair reaction was carried out with substrate containing a G:C or G:U bp at position 22 (A) and [P]dNTP (*) as indicated above each lane. Panel C, position of the alpha-P-label incorporated nucleotide in the 51-oligonucleotide product. The repair reaction was conducted in the presence of 10-20 units of various restriction endonucleases, as shown at the top of each lane, except for AccI and XbaI, where the reaction product was phenol extracted and ethanol precipitated before subjecting it to restriction digestion. Numbers on the right show the lower strand size (nts) of the fragment generated by restriction enzyme analysis. The illustration at the top shows the position of [P]dCMP in the different restriction fragments and their respective position and size in reference to the substrate.




Figure 3: Substrate specificity for the incision steps of the repair reaction. Experiments were conducted as described under ``Experimental Procedures,'' and an autoradiogram of typical results is shown. Different substrates containing 5`-end-labeled lower strand were incubated in the standard repair reaction devoid of dNTP and an ATP-regenerating system (ATP, phosphocreatine, and creatine phosphokinase). The base pair corresponding to position 22 is shown at the bottom of each lane, and the substrates are illustrated at the top, as well as the position of the 5`-P(*) label.



To establish the position of [P]dCMP incorporation in the 51-residue product, we carried out restriction endonuclease digestion of the reaction product. In the experiment shown in Fig. 1Bpanel C, BamHI, PstI, or SalI was added at the end of the reaction, while in the cases of AccI or XbaI, the repair reaction product was purified before subjecting it to digestion. Different labeled fragment sizes were generated by the action of each endonuclease. Based on the digestion pattern, it was evident that [P]dCMP was in the 5-residue region between the XbaI and AccI restriction sites (Fig. 1A), presumably at position 22 in the lower strand. The labeled product molecule was not altered by treatment with uracil-DNA glycosylase or heating at 70 °C, as expected (data not shown).

Characterization of the Base Excision Repair Reaction

The repair reaction with the G:U-containing substrate was further characterized by modifying the reaction conditions. As shown in Fig. 2A, eliminating MgCl(2) completely abolished repair activity; some accumulation of a 22-nucleotide intermediate product was observed in the absence of ATP and ATP regenerating system (phosphocreatine and creatine phosphokinase), but minimal amounts of ligated 51 nucleotide product were found; addition of NaCl to 200 mM greatly reduced the activity.


Figure 2: A composite figure showing reaction requirements for base excision repair. The repair assay was carried out as described under ``Experimental Procedures.'' Autoradiograms of typical results are shown. A, modifications. As shown at the top of each lane, the repair reaction was conducted without any modification, in the absence of MgCl(2) or ATP regenerating system (ATP, phosphocreatine, and creatine phosphokinase), and in the presence of 200 mM NaCl. B, analysis of intermediate products formed during the repair reaction. The 5`-end-labeled lower strand containing unlabeled dUMP was incubated in the standard repair reaction devoid of dNTPs and ATP-regenerating system (ATP, phosphocreatine, and creatine phosphokinase). The reaction was carried out for 20 min in the presence and absence of dCTP as shown. C, effect of addition of dideoxy-CTP in the repair reaction. The repair reaction was carried out in the presence of 5`-end-labeled substrate. The lower strand, containing the unlabeled dUMP residue, carried the 5`-end label. The standard repair reaction was carried out in the absence or presence of dCTP and ddCTP, as shown at the top of each lane. The number in the right-hand margin indicates the length (nts) of the radiolabeled products. Whereas NE at the top represents bovine testis nuclear extract.



As omitting ATP probably blocked the repair reaction at the DNA ligase step, we decided to exploit this property to identify products of uracil DNA glycosylase-endonuclease activities, as well as the DNA polymerase activity step. The lower strand of the G:U-containing duplex substrate was 5`-end-labeled, and this substrate was incubated with the extract without other additions, i.e. the reaction mixture did not contain dCTP, ATP, or ATP regenerating systems. Under these conditions, labeled material corresponding to the starting 51-nucleotide molecule was shifted to an oligonucleotide 21 residues long. When dCTP was then added to the reaction mixture, we observed a band corresponding to an oligonucleotide of 22 residues, as shown in Fig. 2B. To determine if the lack of ATP would result in accumulation of the 22 nucleotide long intermediate, we carried out a similar reaction (in the presence of all components) using 5`-end-labeled lower strand of the G:U substrate. As shown in Fig. 2C, in the absence of dCTP, a 21-nucleotide product accumulated that was converted to full-length product in the full repair reaction containing dCTP. When ddCTP was used in place of dCTP, accumulation of a 22-nucleotide product was observed, as expected for a beta-pol-mediated reaction.

Specificity of the Excision Reaction

We found that a molecule migrating in the gel as a 21-nucleotide oligomer is an intermediate in the overall repair reaction. This intermediate could be readily observed by conducting the reaction in the absence of dCTP. To examine whether the phosphodiester backbone incision reaction producing this 21-nucleotide intermediate was specific to a uracil-containing substrate, we designed different combinations of base pairs at position 22 by altering the lower and upper strand of the 51-bp oligonucleotide (Fig. 1A), i.e. A:C, A:U, A:T, G:C, G:U, and G:T. The lower strand of all these substrates was 5`-end-labeled. Reactions were carried out in the absence of dCTP, ATP, and ATP regenerating systems (phosphocreatine and creatine phosphokinase). As shown in Fig. 3, use of only the A:U- and G:U-containing substrates gave rise to the intermediate product molecule of 21 nucleotides, indicating that our repair reaction was specific for the uracil base. The absence of the intermediate product of 21 nt in the case of G:T and other mismatch substrates may be attributed to relatively short incubation times used for uracil-initiated BER, thus, distinguishing this reaction from that of G:T mismatch repair reaction described by Wiebauer and Jiricny(9) . Use of substrates with a 5`-end-labeled upper strand, instead of lower strand, failed to result in any shorter products.

Inhibition of the Repair Reaction

Results of Fig. 2and Fig. 3indicated that the uracil base was selectively removed and replaced by the base complimentary to the template G, and then the strand was sealed by DNA ligase to generate the full-length 51-bp product. The fact that ddCMP was incorporated and accumulated suggested that beta-pol was involved in the repair reaction. To initially examine the DNA polymerase requirement, we used the inhibitors ddCTP and aphidicolin. As shown in Fig. 4, ddCTP completely inhibited the repair reaction, whereas aphidicolin did not have a noticeable effect (in an experiment not shown, neutralizing antibodies against alpha-pol did not inhibit the repair reaction), suggesting that beta-pol, but not alpha-pol was involved. We also carried out the repair reaction by preincubating the nuclear extract separately with preimmune serum or with polyclonal antibodies raised against native beta-pol and its 8 kDa domain, respectively. Preimmune serum had no effect, whereas the antibodies raised against beta-pol or the 8 kDa domain completely inhibited the repair reaction (Fig. 4A). These two polyclonal antibodies, which were raised and purified in the current study, gave a positive signal on immunoblotting with purified beta-pol, bovine testis nuclear extract, and S. cerevisiae DNA pol IV, a homolog of beta-pol (S. cerevisiae beta-pol); these antibodies did not cross-react with PCNA, human DNA polymerases, alpha, , and , or with E. coli pol I (Fig. 4B). When preincubated with purified beta-pol, both of these new antibodies completely inhibited beta-pol enzymatic activity (Fig. 4C) but did not alter activity of the other DNA polymerases (data not shown).


Figure 4: A composite figure showing inhibition of the repair reaction. A, as indicated at the top of each lane, the standard repair reaction was carried out in the presence of bovine testis nuclear extract alone (none) or in the presence of a 100-fold molar excess of ddCTP over dCTP or 5 µg/ml aphidicolin. Additionally, the nuclear extract was mixed (1:1 volume) with preimmune IgG or polyclonal antibody (IgG) raised against purified rat beta-pol or its 8 kDa domain. The proteins were preincubated at 0-1 °C for 45 min. The standard repair reaction as indicated was carried out. The position of the product is indicated by arrow. B, characterization of the anti-beta-pol polyclonal antibodies. Western blot of S. cerevisiae beta-pol (2 µg) Escherichia coli pol I (0.5 µg), PCNA (2 µg), -pol (2 µg), -pol (2 µg), alpha-pol (0.5 µg), rat beta-pol (150 ng), and bovine testis nuclear extract (150 µg) was conducted using preimmune serum and antibodies (as shown above) as described under ``Experimental Procedures.'' C, the effect of preimmune serum and antibodies were tested on beta-pol gap filling activity, as described under ``Experimental Procedures'' and indicated at the top of each lane.



Separation of DNA Repair Activities on Superose 12 FPLC Column

Bovine testis crude nuclear extract was passed though Mono Q and Mono S columns connected in tandem. The Mono S column was separated and eluted with 1 M KCl, and the eluate was concentrated before loading it onto a Superose 12 gel filtration column pre-equilibrated with buffer containing 1 M KCl to minimize protein-protein interaction (complex formation). The protein elution profile of the Superose 12 column is shown in Fig. 5A. Individual fractions were subjected to various enzymatic assays. DNA ligase activity eluted near the void volume and peaked in fraction 28 as judged by the formation of 73-mer ligase product (Fig. 5B). beta-Pol was the next activity to emerge from the column with maximum 5-nucleotide gap-filling activity in fraction 31, corresponding to the 39-kDa beta-pol monomer. To monitor uracil DNA glycosylase/AP endonuclease activities, the duplex substrate was 5`-end-labeled in the uracil-containing strand, and the reaction was carried out in the absence of dCTP, ATP, phosphocreatine, and creatine phosphokinase. Accumulation of a 21-residue product was maximal in fraction 33 (Fig. 5B).


Figure 5: A, fractionation of the base excision repair reaction proteins by gel filtration column chromatography. As described under ``Experimental Procedures,'' approximately 100 mg of bovine testis crude nuclear extract was loaded onto Mono Q and Mono S columns connected in tandem. The Mono S column was eluted with 1 M KCl, and the proteins were concentrated in Centricon-10 concentration units. A, absorbance (280) elution profile of FPLC Superose 12 HR 10/30 gel filtration column. A sample, 200 µl (approx300 µg of protein) of concentrated 1 M KCl fraction of Mono S column, was injected onto Superose S 12 column, and 0.5-ml fractions were collected. Respective positions of the molecular weight markers are indicated in the figure. B, assay of different enzymatic activities in the column fractions. All the fractions were concentrated to approx 100 µl in buffer C. An equal volume (10 µl) of fraction sample was used in each enzymatic activity assay. DNA ligase and DNA polymerase beta activities were determined as described under ``Experimental Procedures.'' The DNA ligase end product (73-mer) resulted after ligation of the beta-pol gap filling reaction product (22-mer) with 51 nt oligo annealed down stream to the M13 primer to create a 5-nt gap. Glycosylase and endonuclease activities were detected by using a G:U containing substrate with 5`-end-labeled U-containing strand. The standard repair reaction was carried out in the absence of dNTP, ATP, phosphocreatine, and creatine phosphokinase. A standard repair assay was also conducted on the Mono S column fraction (lane S) that was loaded on to the Superose 12 column. Enzymatic activities, their respective products, and the fractions tested are shown in the figure.



Fractions also were assayed for overall G:U repair activity. Product accumulation, corresponding to the 22-residue molecule, but not the fully repaired 51-residue molecule, was maximal in fraction 32. Additionally, the original sample applied to the Superose 12 column was found to have base excision repair activity (lane S) as expected. Overall, these results show that several of the enzymatic activities required for the repair reaction could be separated from one another by gel filtration chromatography and that all of the enzymatic activities required were leq50 kDa, except for DNA ligase.

Reconstitution of Base Excision Repair Reaction

We used fraction 36 (Fig. 5B) to reconstitute the repair reaction, as this fraction had uracil DNA glycosylase/AP endonuclease activities, but was devoid of overall repair, DNA polymerase, or DNA ligase activities. As shown in Fig. 6, fraction 36 alone failed to show any overall DNA repair activity, and the addition of T4 DNA ligase had no effect. However, the usual 22-residue intermediate product molecule was formed when purified beta-pol was added. Formation of this product was not observed when the 31 kDa domain of beta-pol was added instead of intact beta-pol (data not shown). Next, we found that the 22-residue product was shifted to the full-length product (51-mer), when both beta-pol and DNA ligase were added. Formation of the 22-residue intermediate product by fraction 32 was abolished by polyclonal antibody against beta-pol or its 8 kDa domain. Finally, the presence of beta-pol in fraction 32 was confirmed by immunoblotting; this experiment confirmed the presence of the 39-kDa enzyme (data not shown).


Figure 6: Reconstitution of the repair reaction and its inhibition by beta-pol antibodies. Fraction numbers 36 and 32 of the Superose 12 gel filtration column were assayed for base excision repair. T4 DNA ligase (3 µg), purified recombinant beta-pol (0.4 µg), and both T4 ligase and beta-pol together were added in the repair reaction with fraction 36, while only ligase was with fraction 32. Inhibition of the repair activity was tested by addition of fraction 32 preincubated on ice (1:1 volume) with preimmune serum and the polyclonal antibodies raised against beta-pol and its 8-kDa domain as shown in the figure.



Reconstitution of the Base Excision Reactions with Different DNA Polymerases

Our results indicated that purified beta-pol can reconstitute the BER reaction in vitro and that the endogenous polymerase in fraction 32 is beta-pol, as the enzyme is a 39-kDa DNA polymerase inhibited by specific anti-beta-pol antibodies. To determine if other purified mammalian DNA polymerases were capable of conducting a single nucleotide gap filling reaction, we studied highly purified human DNA polymerases alpha, , and using fraction 36, as in Fig. 6. We found that only beta-pol could reconstitute the full BER reaction; a very minor activity was observed for DNA polymerase alpha, in the presence of a large excess of enzyme (Fig. 7A), and no activity was found with polymerases and .


Figure 7: Reconstitution of the repair reaction with different DNA polymerases. A, fraction number 36 of the Superose 12 gel filtration column was used to conduct the repair reaction. Purified DNA polymerases beta (0.4 µg), alpha (1.5 µg), (0.89 µg), and (2.9 µg) were used as indicated in the figure. Polymerase S activity was measured in the presence of 3 µg of PCNA. B, primer extension activity of the various DNA polymerases were tested by annealing M13mp18 (+) ssDNA template and 17-mer universal primer as shown in the figure. Identical reaction conditions and polymerase concentrations were used as above except that 40 µM unlabeled dATP, dGTP, and dTTP were included.



To establish that the DNA polymerases used in the reactions were active, we carried out a primer extension assay under conditions identical to those used for the repair reaction. As shown in Fig. 7B, all the polymerases tested showed abundant activity, but the amount of beta-pol used in these experiments was much less than for the other polymerases. Although the amount of polymerases used was different, a comparison of the ratio/repair activity to primer extension activity, indicated that beta-pol is far more active in the base excision repair reaction than is alpha-pol.

Uracil Base Excision Repair in S. cerevisiae

A study of uracil and osmium tetroxide-initiated BER in vitro in S. cerevisiae extract implicated DNA polymerase , and the reaction could be influenced by DNA polymerases alpha and (14) . Since the presence of beta-pol-like enzyme was recently demonstrated in S. cerevisiae(15, 16) , we tested the role of purified S. cerevisiae beta-pol in our mammalian BER system. As shown in Fig. 8A, purified S. cerevisiae beta-pol was able to reconstitute polymerase activity in fraction 36 (as in Fig. 6), and the catalytic activity was neutralized by anti-rat beta-pol antibody; this was expected as anti-rat beta-pol antibody cross-reacts with S. cerevisiae beta-pol and also inhibits its gap-filling activity (data not shown).


Figure 8: Uracil base excision repair reaction in S. cerevisiae. A, reconstitution of the repair reaction was carried out as described in Fig. 6except that 0.1 µg of purified S. cerevisiae beta-pol was used. Equal volumes of S. cerevisiae beta-pol and anti-rat beta-pol antibody were preincubated before carrying out the repair reaction as shown in the figure. B, the repair reaction was carried out as described under ``Experimental Procedures.'' Nuclear extract (25 µg) from either wild type strain or beta-pol gene deletion strain was used. Nuclear extract from wild type strain was preincubated with equal volume of preimmune and anti-rat beta-pol antibody as shown in the figure.



Next, we prepared nuclear extract from the S. cerevisiae strain LP3041-6D (wild type) and the strain JWY355 carrying a beta-pol gene deletion, as described under ``Experimental Procedures.'' The uracil-initiated BER reaction, as reported by Wang et al. (14) , was conducted with nuclear extract from both strains (Fig. 8B). In both cases, the 22-residue intermediate product and a small amount of the completely repaired 51-residue product were formed. Most of the 22-mer product could be converted to 51-mer produced by addition of T4 DNA ligase (data not shown), suggesting either a low level of DNA ligase or partial inhibition of ligase activity in the nuclear extracts. Formation of these products was not inhibited by anti-beta-pol antibodies. In an experiment not shown, product formation was not inhibited by ddNTP. Further, addition of purified S. cerevisiae beta-pol had only marginal affect (data not shown). These results corroborate the results of Wang et al.(14) and suggest a different DNA polymerase requirement for uracil-initiated BER in S. cerevisiae than in mammalian cells.


DISCUSSION

Studies to understand the DNA polymerase requirement(s) for various eukaryotic excision repair mechanisms have been under way for a number of years and have led to the understanding that for gap-filling DNA synthesis, the process can be loosely categorized by repair gap or patch size as follows(30) : long-patch repair (>50 nt) as in mismatch repair; intermediate-patch repair (24-50 nt) as in nucleotide excision repair; and short-patch repair (leq3-4 nt) as in base excision repair. Two studies of BER in vitro, involving short-patch repair (9, 11) , have pointed to a requirement for beta-pol, and the enzyme has been clearly demonstrated to have the capacity to conduct short-patch gap filling in vitro(17, 18, 19) . Our results established that beta-pol is responsible for the single-nucleotide DNA synthesis step involved. Multiple observations indicated that the DNA strand containing uracil was repaired via a single-nucleotide excision gap: 1) dCTP alone was sufficient to support the reaction; 2) the product of the endogenous endonuclease incision step was only one nucleotide shorter (i.e. 21 nt) than the position of uracil residue; 3) addition of pure beta-pol extended the 21-nt intermediate product molecule by only one nucleotide; 4) the 22-nucleotide intermediate product accumulated in the reaction mixture when ATP was omitted to intentionally block the activity of DNA ligase; 5) and finally, the DNA synthesis reaction was completely blocked by two specific, neutralizing polyclonal antibodies to beta-pol. Four of the major enzymatic activities required for the reaction, DNA ligase, DNA polymerase, uracil-DNA glycosylase, and endonuclease(s), could be partially resolved by gel filtration chromatography using a Superose-12 column. DNA ligase activity eluted at a higher molecular mass than the other activities, all of which had molecular mass of leq50 kDa. Once ligase activity had been removed, addition of the DNA ligase-containing fraction to the depleted extract only partially restored the BER activity (data not shown). The requirement for DNA ligase, however, could be fully complemented by addition of purified T4 DNA ligase. In a similar fashion, activities providing uracil DNA glycosylase and endonuclease (processing of the AP site) could be purified free of the 40-kDa DNA polymerase activity (i.e. beta-pol), and addition of purified beta-pol was capable of restoring full repair activity. Furthermore, among the four different purified mammalian DNA polymerases tested here, DNA polymerase beta was the only enzyme capable of restoring full repair activity. In these experiments with polymerase-depleted fractions, DNA polymerase alpha was able to minimally reconstitute activity but this required a very high enzyme concentration. Neither aphidicolin nor neutralizing antibody to alpha-pol had a blocking effect with the crude extract system. In a related study (data not shown), addition of purified DNA polymerase beta to HeLa cell nuclear extract resulted in 5-10-fold increase in the uracil-initiated BER whereas DNA polymerase alpha, , and did not, suggesting a role for beta-pol.

Our results with the S. cerevisiae BER in vitro system corroborate the results of Wang et al.(14) . We conclude there was no apparent requirement for beta-pol, since extract from the beta-pol deletion strain was fully active in BER. In addition, the S. cerevisiae BER reaction was not inhibited by ddNTP or neutralizing antibody to beta-pol. These results indicate that the S. cerevisiae and mammalian systems for BER under study here appear to have different DNA polymerase requirements.

In conclusion, we have demonstrated by several criteria that the uracil-initiated BER DNA polymerase activity in our crude extract is beta-pol. These criteria include: its small size, inhibition of activity by neutralizing polyclonal antibodies against beta-pol, complete inhibition by dideoxynucleotide, and by reconstitution of the BER activity using purified beta-pol. Interestingly, a recent study (31) indicating embryonic lethality caused by a null mutation of the beta-pol gene, along with the results of Wang et al.(32) and Sadakane et al.(33) showing novel alterations in beta-pol mRNA in colorectal cancers and in Werner syndrome cells, respectively, raise the possibility of a role of beta-pol in these diseases. The multiple enzymatic activities in the lower molecular weight fractions from our gel filtration column, which contain uracil-DNA glycosylase and endonuclease, have not been resolved. There is not yet a consensus in the literature, as to the involvement of AP endonucleases alone, AP endonuclease and 5`3` exonuclease, AP endonuclease and deoxyribophosphodiesterase (dRpase), or other cellular protein (for discussion, see (11) ), together with uracil-DNA glycosylase to create the single nucleotide gap.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1068. Tel.: 409-772-3367; Fax: 409-772-6334.

(^1)
The abbreviations used are: BER, base excision repair; pol, polymerase; AP, apurinic/apyrimidinic; PCNA, proliferating cell nuclear antigen; ss, single-stranded; FPLC, fast protein liquid chromatography; bp, base pair(s); nt, nucleotide(s).


ACKNOWLEDGEMENTS

We are indebted to Dr. M. Lee for providing purified PCNA and DNA polymerase and , and we thank Drs. L. Prakash, S. Lloyd, S. Mitra, and B. Van Houten for their valuable suggestions and critical reading of the manuscript.

Addendum-DNA polymerase beta is able to conduct repair of only natural AP sites in abasic site repair in X. laevis oocytes(34) .


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