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J. Biol. Chem., Vol. 277, Issue 21, 18454-18458, May 24, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18454    most recent M200421200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ramadan, K. Articles by Hübscher, U. Search for Related Content PubMed PubMed Citation Articles by Ramadan, K. Articles by Hübscher, U. Social Bookmarking                      What's this?

DNA Polymerase  from Calf Thymus Preferentially Replicates Damaged DNA^*

Kristijan Ramadan, Igor V. Shevelev^§, Giovanni Maga^¶, and Ulrich Hübscher

From the  Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland, ^§ Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Leningrad district, Gatchina, 188300, Russia, and ^¶ Istituto di Genetica Molecolare, Consislio Nationale dell Ricerche, Via Abbiategrasso 207, I-27100, Pavia, Italy

Received for publication, January 15, 2002, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

A new gene (POLL), has been identified encoding the novel DNA polymerase  and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase  contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase , , µ, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase  from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase  was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase  and polyclonal antibodies against DNA polymerase . DNA polymerase  had no detectable nuclease activities and, in contrast to DNA polymerase , was aphidicolin-sensitive. DNA polymerase  was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase . The biochemical properties of the calf thymus DNA polymerase , described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Eukaryotic cells contain several DNA polymerases (pol)¹ classified as classical pols (, , , , and ) and novel pols (, , , , , , , and µ) (1). Novel pols have so far never been purified directly from mammalian tissues.

Recombinant murine pol  (67 kDa) is a protein composed of 573 amino acids and is highly similar to the members of the pol family X (2) comprising enzymes involved in the DNA repair processes, whose main member is pol . The first 239 amino acids of the N-terminal part of pol  with the exception of the nuclear localization signal have no counterpart in pol . This N-terminal part of pol  has similarity to yeast pol IV and contains a BRCA1 C-terminal (BRCT) domain (2). The BRCT domain is present in several proteins involved in DNA repair and cell cycle checkpoint control (3, 4). Recently, it has been shown that the BRCT domain is involved in protein/protein interactions (4). The remaining part of pol  is composed of the catalytic core, which is similar to pol  (8-kDa DNA deoxyribophosphodiesterase domain and 32-kDa finger, palm, and thumb-polymerization domain) and has 32% amino acid identity to pol  (2).

Pol  contains 5'-deoxyribose phosphate lyase activity, but no apurinic lyase activity (5), and can substitute pol  in in vitro base excision repair (BER), suggesting that pol  participates in BER. Pol  is the main pol involved in the BER of lesions generated by monofunctional alkylating agents in eukaryotic nuclear DNA, and pol  was proposed to be involved in the other types of BER (5). However, understanding the possible physiological roles of this enigmatic pol would require a complete characterization of the biochemical properties of the endogenous enzyme. In this study, we describe, for the first time, the isolation of endogenous pol  from a mammalian tissue. Remarkably, this activity was isolated by an activity assay that uses damaged DNA as a template, suggesting a possible role of pol  in DNA repair and translesion synthesis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Chromatographic Media and Other Chemicals-- Phosphocellulose P11 was obtained from Whatman, hydroxyapatite from Bio-Rad, HiTrap Desalting, HiTrap heparin, mono Q, and mono S columns from Amersham Biosciences. Pepstatin, leupeptin, and aphidicolin were obtained from Sigma. All other reagents were of analytical grade and purchased from Merck or Fluka.

Buffers-- All stock solutions were filtered through nitrocellulose before use (0.45 µm, Schleicher & Schüll). The following buffers were used: buffer 1 (50 mM Tris-HCl, pH 7.0, 50 mM NaCl, 20% (w/v) sucrose, 1 mM DTT, 2 mM EDTA, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 0.8 mM PMSF); buffer 2 (50 mM Tris-HCl, pH 7.0, 50 mM NaCl, 1% (v/v) Triton X-100, 1 mM DTT, 2 mM EDTA, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 0.8 mM PMSF); buffer 3 (50 mM Tris-HCl, pH 7.0, 1 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 0.8 mM PMSF, and different molar concentrations of NaCl), buffer 4 (50 mM Tris-HCl, pH 7.0, 1 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 0.8 mM PMSF, and different molar concentrations of potassium phosphate); buffer 5 (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 0.8 mM PMSF, and different molar concentrations of NaCl).

Preparation of Nucleic Acids-- Amersham Biosciences was the supplier of poly(dA) and oligo(dT)_12-18. The homopolymer poly(dA) was mixed to the oligomer oligo(dT)_12-18 at a 10:1 base ratio in 20 mM Tris, pH 8.0, 20 mM KCl, and 1 mM EDTA heated at 60 °C for 5 min with subsequent slow cooling to room temperature.

Damaged (Apurinic) DNA Template Preparation and Characterization-- To create apurinic sites, the DNA template poly d(A)/oligo d(T) at a 10:1 base ratio was incubated at a final concentration of 0.25 mg/ml at 70 °C in the following buffer: 10 mM sodium citrate, 10 mM NaCl, and 10 mM NaH₂PO₄, pH 5.2. This treatment specifically hydrolyzes the glycosidic bond between the purine base and the deoxyribose moiety, releasing the base without interrupting the continuity of the sugar-phosphate backbone. The degree of DNA damage was checked at different incubation times by monitoring the loss of DNA synthesis efficiency by pols , , and . For the preparation of the apurinic poly(dA)/oligo(dT) template, an incubation time of 40 min was selected, corresponding to a loss of 80% DNA synthesis efficiency by pols  and  and 73% by pol  if compared with the corresponding non-damaged poly(dA)/oligo(dT).

Antibodies-- The mouse monoclonal antibody against rat pol  protein was purchased from NeoMarkers (Freemont, CA). The rabbit polyclonal antibody against mouse pol  was a gift from L. Blanco (Madrid, Spain).

DNA Polymerase Assays-- A final volume of 25 µl contained the following components: 50 mM bis-Tris, pH 6.5, 1 mM DTT, 250 µg/ml bovine serum albumin, 6 mM MgCl₂, 10 mM KCl, 20 µM [³H]dTTP, 0.5 µg of poly(dA)/oligo(dT)_12-18 (10:1 base ratio) or 0.1 µg of depurinated poly(dA)/oligo(dT)_12-18 (10: 1 base ratio) and enzyme to be tested. 1 unit of enzyme activity corresponds to the incorporation of 1 nmol of dTTP into acid-precipitable material in 60 min at 37 °C.

Purification of a DNA Polymerase Activity That Preferentially Uses a Damaged DNA Template-- All enzymatic pol tests were performed with poly d(A)/oligo (dT)_12-18 or with damaged poly d(A)/oligo (dT)_12-18, and isolation steps were performed at or near 0 °C. 70 g of calf thymus were resuspended in 210 ml of buffer 1 and homogenized in a Sorvall Omnimixer. After centrifugation in a GSA rotor at 12,000 rpm for 45 min, the pellet was washed additionally in buffer 1. This pellet (35 g) was resuspended in 140 ml of buffer 2, homogenized, rotated for 2 h, and centrifuged at 12,000 rpm for 45 min. The supernatant (detergent extract) was brought to pH 7.0 to yield fraction I. Fraction I was loaded onto a 60-ml P11 phosphocellulose column, equilibrated previously in buffer 3 with 50 mM NaCl. The column was first washed with 600 ml of buffer 3 (50 mM NaCl), and the proteins were eluted with 500 ml of a linear 50-700 mM gradient of NaCl in buffer 3. The peak fractions containing pol activity on damaged DNA eluted between 220 and 300 mM NaCl and were pooled to yield fraction II. Fraction II was adsorbed to a 6-ml hydroxyapatite column, equilibrated previously in buffer 4. After washing with 10 column volumes of buffer 4 with 20 mM potassium phosphate, a 10-column volume of a linear 20-500 mM potassium phosphate gradient in buffer 4 was developed. On this column, separation of pols that do not synthesize on the damaged template was achieved (see Fig. 1A). The pol activity on the damaged template was eluted between 250 and 350 mM potassium phosphate, pooled, and desalted to yield fraction III. Fraction III was loaded on a 1-ml HiTrap heparin column equilibrated in buffer 3 (50 mM NaCl), the column was washed with 6 ml of buffer 3 (50 mM NaCl), and the pol activity was eluted with a 40-ml linear gradient of 50-1000 mM NaCl in buffer 3. The active fractions were eluted between 550 and 650 mM NaCl, pooled, and desalted to yield fraction IV. Fraction IV was loaded onto a fast protein liquid chromatography mono Q column, equilibrated in buffer 5 (50 mM NaCl), washed with 5 ml of buffer 5 (50 mM NaCl), and eluted with a 15-ml 50-1000 mM NaCl linear gradient in buffer 5. The active fractions were eluted between 380 and 500 mM NaCl, pooled, and desalted to yield fraction V. Fraction V was loaded onto a fast protein liquid chromatography mono S column, equilibrated in buffer 5 (50 mM), and washed with 5 ml of buffer 5 (50 mM NaCl). The pol activity was eluted with a 10-ml 50-500 mM linear gradient in buffer 5 as a homogeneous peak. The active fractions were immediately frozen in small aliquots to 80 °C until further use.

Fidelity Assay-- Bacteriophage M13mp2 and Escherichia coli strains CSH50, NR9099, and MC1061 were as described in Ref. 6. Nonsense mutant M13mp2T90 (point mutation G:T in the position +90 of the lacZ gene) was selected after sequencing analysis of colorless mutants as a mutation hot point in the DNA M13mp forward mutation assay. The sequence of the 26-mer oligonucleotide was: 5'-cga tta agt tgg gta acg cca ggg tt-3'. This oligonucleotide was hybridized in addition to the gap molecules (the 3'-end of the primer being located as three nucleotides from the point substitution T in the nonsense mutant instead of G in M13mp2 DNA at position +90 of lacZ gene) to verify that in every case, the nucleotides were incorporated in the nonsense codon TAA. Forward and reverse mutation assays were done according to Ref. 6. Replication reactions for the forward mutation assay were carried out for 2 h at 37 °C in 10 µl containing: 5 mM MgCl_2, 50 mM Tris-HCl, pH 7.2, 1 mM DTT, 100 µg/ml bovine serum albumin, 40 ng of gapped circular M13mp2 DNA, 20 µM each of dATP, dCTP, dGTP, and dTTP, and 0.03 units of either pol  or pol . In this case, about 250-300 nucleotides were incorporated per DNA template, although for calculation of the error rate, it was necessary to fill 250 nucleotides of 350 gapped. Replication reactions for the reverse mutation assay were carried out for 10 min by using as a template M13mp2T90 DNA with an additional 26-mer oligonucleotide. All other conditions were identical to the forward mutation assay.

Other Methods-- The following three methods were carried out according to established protocols: SDS-PAGE (7), immunoblotting (8), and in situ polymerase activity gel analysis (9).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Purification of a 67-kDa DNA Polymerase from Calf Thymus Detergent Extract That Preferentially Replicates Damaged DNA-- Calf thymus detergent extract was fractionated using a damaged DNA as the template for measuring DNA polymerase activity (see "Materials and Methods"), and DNA polymerase activity was monitored through different chromatographic steps (Fig. 1A). On the second purification step, a peak eluting between 250 and 350 mM potassium phosphate, which had a similar activity on both the non-damaged and damaged DNA, was separated by the bulk of DNA polymerase activity (Fig. 1B). This activity was then purified through HiTrap Heparin-Sepharose, mono Q and mono S columns. The resulting peak fractions (Fig. 1C) were analyzed on SDS-PAGE and showed a single band of 67 kDa (Fig. 1D).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Purification of a 67-kDa DNA polymerase from calf thymus detergent extract that preferentially replicates damaged DNA. A, flow chart of the purification procedure (for details, see "Materials and Methods"). F, fraction. B, chromatographic separation of a pol that is preferentially active on damaged DNA from the classical pols. The second, minor peak (arrow, eluting between 250 and 350 mM potassium phosphate) shows similar pol activity on non-damaged DNA and on damaged DNA. The specific activities were 7.6 units/mg on non-damaged DNA and 5.1 units/mg on damaged DNA. The pooled fractions 12-17 were used for further purification. C, the last chromatography step, the mono S column. The pol activity eluted between 350 and 300 mM NaCl on mono S column as one peak. The pol activity was 2064 units/mg on damaged DNA versus 1040 units/mg on non-damaged DNA (details of chromatographic behavior are described under "Materials and Methods"). D, fractions V and VI analyzed in a 12.5% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue. Only one band at a position of 67 kDa was identified in fraction VI. MWM, molecular weight markers.

Identification of the 67-kDa DNA Polymerase as DNA Polymerase -- The final mono S fraction (fraction VI) was analyzed by immunoblot for a variety of known pols, such as pols , , , and . No bands were observed with antibodies against pols , , and  (data not shown), but a strong signal was discovered for a 67-kDa polypeptide with a monoclonal antibody against human recombinant pol  (Fig. 2A), suggesting that we had isolated a -like pol. Next, the same mono S fraction was tested with an antibody against pol , and a clear signal was again obtained for this polypeptide (Fig. 2B). All pols of family X have very conserved DNA polymerase domains (2); therefore, the cross-reactivity of pol  with pol  antibody was not unexpected. Next, we tested whether this 67-kDa polypeptide was responsible for the polymerase activity. As shown in Fig. 2C, in situ activity gel analysis showed that the polypeptide corresponding to the 67-kDa band was able to incorporate nucleotides into a DNA template. These data strongly suggested that the pol isolated from calf thymus using a damaged DNA as the template is pol . Accordingly, only pol  among family X pols has an expected molecular mass of 64-67 kDa based on the cDNA sequence of human and mouse.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Identification of the 67-kDa polypeptide as pol . A, fractions (F) V and VI (see panel A of Fig. 1) immunoblotted after 12.5% SDS-polyacrylamide gel electrophoresis with monoclonal antibodies against pol . The pol  antibody cross-reacted with a band at position 67 kDa in both fractions. The recombinant pol  was used as a positive control. MWM, molecular weight markers. B, immunoblot analysis for pol . Polyclonal antibodies against pol  cross-reacted with a 67-kDa polypeptide in both fractions V and VI. C, activity gel analysis performed to confirm the pol activity in the 67-kDa polypeptide. Fraction VI and pol  were tested in a 12% SDS-PAGE activity gel analysis as described under "Materials and Methods."

It has been reported that N-terminal domain of pol  shares similarity with yeast pol IV and that the C-terminal domain shares similarity with mammalian pol  (2). Although yeast pol IV shows the properties typical for pol , the apparent molecular mass of the polypeptide is larger (68 kDa) (10). Therefore, it is possible that pol , rather than pol , is the homolog of yeast pol IV.

Biochemical Properties of DNA Polymerase -- Since the pol activity after the last mono S column was low, an optimization and characterization was performed. These are summarized in Fig. 3. The pH optimum of pol  was 6.5 with 50 mM bis-Tris, Tris-HCl, or 15 mM potassium phosphate buffers. The Mg²⁺ optimum was 1.5 mM, and for Mn²⁺, it was 0.5 mM. The K_m for the nucleotide substrate (dTTP) was 5.9 µM, and the V_max was 0.38 pmol/min. The ratio V_max/K_m was 2.57 × 10³ min¹ × 1 µl¹, which is a lower estimation of k_cat/K_m. These results are significantly different from those for human recombinant pol , which has low K_m (0.5 µM) for the nucleotide substrate and optimal conditions for pol activity at pH 7.5 and 10 mM MgCl₂ (11). Pol  is resistant to inhibition by aphidicolin (10, 12); therefore, it was interesting to examine the purified calf thymus pol  for this inhibitor. Unexpectedly and in contrast to pol , pol  was aphidicolin-sensitive.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Biochemical properties of calf thymus DNA polymerase . All pol  assays were performed as described under "Materials and Methods." 100% of activity corresponds to 21 units/ml on damaged DNA. A, dependence of activity on divalent cations. F, fraction. B, dependence of the activity on the pH in different buffer systems. C, sensitivity of polymerase activity to aphidicolin. DNA pol  was inhibited by aphidicolin (big squares). DNA pol  was used in the same experiment as a control for aphidicolin resistance (small squares). D, dependence of reaction velocity on dTTP concentration. Curves were fitted to the experimental points by non-linear least squares fitting.

Fidelity of DNA Polymerases  and  in a Forward and in a Reverse Mutation Assay-- Pol  is the least accurate classical mammalian pol, with an average substitution error rate of about 10³ (13). Human recombinant pol µ (a member of family X DNA polymerases) is an error-prone pol (14). Replication fidelity of natural pol  has never been measured before. Therefore, we compared the fidelity of calf thymus pol  with human recombinant pol . A 6-fold more accurate synthesis was observed for pol  as compared with pol  in a forward mutation assay (Table I) when both base substitution and frameshift mutation were determined. We have used 2-h gap filling reactions. Under these conditions, even if neither a 3'5' exonuclease nor any other nuclease was identified in pol  fractions, a very minor proofreading activity could influence the overall fidelity due to a long incubation. To exclude this probability and estimate a share of base substitutions from total errors, we used the additional reverse mutation system on M13mp2T90 DNA with only a 10-min synthesis (see "Materials and Methods"). The data from Table II confirmed a 5-fold higher fidelity of pol  as compared with pol . Errors can occur during any DNA synthesis reaction in a cell, including the gap filling synthesis required for mismatch repair, BER, or nucleotide excision repair. These repair processes require different amounts of DNA resynthesis, catalyzed by pols having very different error rates. The main repair pol  has an average substitution error rate of about 10³. If this was the error rate in vivo, only BER-associated synthesis of pol  would yield about 10 errors per day (15). However, the fidelity of a complete BER complex must be higher than that of the pol alone. It was shown that pol  misinsertion might be proofread by an extrinsic exonuclease (16, 17) or subsequently corrected by some forms of DNA mismatch repair. Furthermore, it was shown that an alternative BER pathway can utilize pols  and . Using nuclear extracts from wild-type and -pol null mouse fibroblasts, it has been demonstrated that this alternative BER pathway has a repair patch size of about two to six nucleotides (18). We have identified pol  from calf thymus as a pol activity scoring better on damaged versus non-damaged DNA. The facts that pol  can incorporate nucleotides on damaged DNA and has 5- to 6-fold higher fidelity than pol  suggest that pol  can contribute substantially to DNA repair. Thus, based on our results, we suggest that pol  could be responsible for this additional long patch BER pathway. It was shown recently that human recombinant pol  performs a limited but significant strand displacement synthesis on gapped DNA substrates, a capacity that would be essential to allow the participation of pol  in long patch BER (5). It was also shown that human recombinant pol  exhibits 5'-deoxyribose phosphate lyase activity and, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. Thus, pol  may participate in single-nucleotide BER in mammalian cells. The mRNA for human pol  was highly abundant in testis, although basal levels were detected in all tissues examined (2). Therefore, and in addition to demonstrating its putative role in meiotic recombination, it will be relevant to determine whether pol  could also have a role in homologous recombination (e.g. repair of double-stranded breaks, a process contributing to genetic stability in somatic cells).

View this table: [in this window] [in a new window]   Table I Mutation frequencies of DNA polymerases  and  by the forward mutation assay

View this table: [in this window] [in a new window]   Table II Mutation frequencies of DNA polymerases  and  by the reverse mutation assay

	ACKNOWLEDGEMENTS

We thank L. Blanco for polyclonal antibodies against mouse pol  and for critical reading of the manuscript and Graziella Pedrazzi for help in preparing the figures.

	FOOTNOTES

^* This work was supported by Grant 3100. 061361. 00 from the Swiss National Science Foundation and by the Kanton of Zürich.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.

To whom correspondence should be addressed: Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. Tel.: 41-1-635-54-72; Fax: 41-1-635-68-40; E-mail: hubscher@vetbio.unizh.ch.

Published, JBC Papers in Press, March 8, 2002, DOI 10.1074/jbc.M200421200

	ABBREVIATIONS

The abbreviations used are: pol, polymerase; BER, base excision repair; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18454    most recent M200421200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ramadan, K. Articles by Hübscher, U. Search for Related Content PubMed PubMed Citation Articles by Ramadan, K. Articles by Hübscher, U. Social Bookmarking                      What's this?