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polymerase activity by the 14 kDa domain appears ..... At 15 ,uM dTTP, no inhibition was observed at low ..... 9 Dianov, G., Price, A. and Lindahl, T. (1992) Mol.
Nucleic Acids Research, 1995, Vol. 23, No. 9 1597-1603

Specific inhibition of DNA polymerase by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition Intisar Husain, Bradley S. Morton, William A. Beard1, Rakesh K. Singhal1, Rajendra Prasad1, Samuel H. Wilson1 and Jeffrey M. Besterman* Department of Cell Biology, Glaxo Research Institute, Research Triangle Park, NC 27709, USA and 1Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555-1068, USA Recieved November 30, 1994; Revised and Accepted March 20, 1995

INTRODUCTION

chemotherapeutic agents may be a DNA repair capacity (4). Specific inhibitors of critical DNA repair enzymes could, therefore, be used to potentiate the cytotoxicity of existing chemotherapeutic agents. Indeed, cells from patients with repair deficiency syndromes are hypersensitive to radiation and various DNA damaging agents (5-7). In base excision repair, mismatch repair, and nucleotide excision repair of DNA damage, the repair process is a sequential multienzyme event (2,3). Following damage and excision, the re-synthesis of the nucleotide sequence is catalyzed by a DNA polymerase before the nick is sealed by DNA ligases. Therefore, DNA polymerases play a critical role in each DNA repair pathway. Mammalian cells contain five known DNA polymerases: a, I, y, 6, E (8). Two mammalian DNA repair systems have been shown to require ,-polymerase for filling short gaps in vitro (9,10) and it has been suggested that ,1-polymerase is involved in repair of the short gaps (i.e., base excision repair) induced by bleomycin and y-radiation (11). In addition, over-expression of n-polymerase has also been implicated in resistance to cisplatin (12-14). However, P-polymerase may also function in DNA replication because it can substitute for Pol I in Escherichia coli and can catalyze the joining of Okazaki fragments (15). As well, [-polymerase is essential for the conversion of single-stranded to double-stranded DNA in Xenopus extracts (16). Since human mutant cell lines deficient in P-polymerase are not yet available, we wanted to define a strategy for determining the role of this enzyme in repair of therapeutically relevant DNA damage. In the past, a number of strategies have been utilized to define the role of 0-polymerase with limited success. For example, the role of [-polymerase during in vivo gap filling synthesis has been defined in intact and permeabilized cells using inhibitors against other cellular polymerases and an inhibitor of

Many cancer chemotherapeutic agents cause direct damage to DNA and this damage is often responsible for the cytotoxicity of these agents. The majority of the DNA lesions are repaired by various DNA repair mechanisms inside the cell (1-3). Unrepaired lesions appear to be responsible for the cytotoxicity and efficacy of chemotherapeutic agents. Moreover, a lack of

Because the specificity of these inhibitors is not absolute, the issue of which DNA polymerase(s) is involved in gap filling in the different DNA repair pathways is not settled. However, DNA polymerase has been clearly identified as the polymerase involved in base excision repair of G:U mismatches (21).

ABSTRACT DNA polymerase (,-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various ,B-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of 0-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits 0-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of ,-polymerase do not inhibit 0-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of P-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of ,-polymerase activity by the 14 kDa domain appears specific to 0-polymerase as this domain does not inhibit either mammalian DNA polymerase a or Escherichia coil polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular P-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.

*

To whom correspondence should be addressed

response to irradiation or consequence of increased

1-polymerase, dideoxynucleoside 5'-triphosphate (11,17-20).

1598 Nucleic Acids Research, 1995, Vol. 23, No. 9 Attempts to reduce the intracellular P-polymerase levels using antisense expression approach have not been fully successful since enzyme levels were only partially depleted (22). In an alternative approach, mutated protein and DNA binding domains have been utilized to inhibit intracellular resident activity of other DNA repair proteins. For example, over-expression of mutated ERCC-1 protein in repair proficient cells competes with the wild type protein in the repair process resulting in a mutated cell phenotype (23). These cells demonstrate higher sensitivity to mitomycin C as compared to wild type cells. In another recent study, introduction of the human poly (ADPR) polymerase DNA binding domain, either as a purified polypeptide or over-expression from an expression vector in transfected cells, selectively interferes and inhibits resident poly (ADPR) polymerase activity (24). This blocking property of the DNA binding domain depends absolutely on binding to DNA breaks through zinc fingers. Therefore, we determined whether a similar approach utilizing domains of ,-polymerase, which lack DNA polymerase activity, could be used to inhibit the activity of this enzyme in vitro. Mammalian DNA polymerase a 39 kDa monomeric protein, exhibits distributive DNA synthesis on a DNA substrate in which a primer is annealed to a single-stranded template and processive gap-filling on short-gapped (up to 6 nt) duplex DNA substrates which have a 5'-phosphate on the downstream oligonucleotide (25). The intact enzyme is capable of binding both single- and double-stranded nucleic acids. The chemical and proteolytic cleavage of 3-polymerase generates domains that are devoid of catalytic activity, but retain DNA binding capacity. Of these domains, only the N-terminal 14 kDa domain binds both singleand double-stranded DNA. Based on these observations, we wanted to know whether the 14 kDa domain could inhibit 0-polymerase activity in vitro. an

j3,

MATERIALS AND METHODS Materials

Deoxynucleoside triphosphates, poly(dA), p(dT)16, and p(dT)20 were purchased from Pharmacia. [a-32P]dTTP (3000 Ci/mmol) was obtained from DuPont/New England Nuclear Corporation. Klenow fragment and bovine serum albumin were purchased from Gibco-BRL. Immobilon-S membrane was obtained from

Millipore (Bedford, MA). The catalytic subunit of a-polymerase, which was expressed in baculovirus and purified, was a generous gift from Dr William Copeland (NIEHS). HPLC purified heteropolymeric oligomers of defined sequence were obtained from Operon Technologies, Inc. T4 polynucleotide kinase was from US Biochemicals and Nensorb-20 columns were obtained from DuPont. DNA polymerase domains The 8 kDa fragment of ,3-polymerase and intact 3-polymerase were overexpressed in E.coli and purified as described earlier (26-28). To facilitate analysis of the 14 and 31 kDa domains, expression plasmids were constructed with the coding sequences of each domain, residues 1-140 and 87-334, respectively. These were overexpressed in E.coli and purified to homogeneity. The 16 kDa domain (residues 18-154) was obtained by CNBr treatment of the holoenzyme (29) and the 27 kDa domain of polymerase

was prepared by chymotrypsin digestion of purified enzyme. Both fragments were purified as described previously (27-29).

DNA polymerase reactions DNA synthesis by [-polymerase was measured with poly(dA)539/p(dT)16 template-primer (T-P). The T-P was constructed by annealing p(dT) 16 to poly(dA)539 at a nucleotide ratio

of 4.2 (template to primer) by heating the mixture to 95°C and slowly cooling to room temperature over several hours to prevent stacking of the oligo(dT) on the template (30). The final reaction mixture contained 50 mM Tris-HCl pH 7.5, 5 mM MnCl2, 25 mM KCl (unless otherwise indicated), 2% glycerol, 5 nM ,3-polymerase, 125 nM dTTP and 5 nM poly(dA)-p(dT)16 (expressed as primer 3'-OH termini) unless otherwise indicated. The concentration of the P-polymerase domains in each reaction is indicated in the figures. Reaction mixtures were incubated at 25'C for 10 min and stopped by adding EDTA to a final concentration of 50 mM. The reaction mixtures were filtered through a manifold with an Immobilon-S membrane. Unincorporated [a-32P]dTTP was removed by five washes of 0.3 M ammonium formate pH 8. The dried filters were cut into individual wells and counted in 3 ml Ecolume scintillation fluid. Alternatively, radioactive products were collected on Whatman DE81 filter disks as described previously (31). The polymerase activity of Klenow fragment was determined as above but with 10 mM MgCl2 or MnCl2. DNA polymerase a activity was also determined as described above. Further details are provided in the figure legends. The apparent equilibrium dissociation constants (i.e., Kd,p) for the binding of heteropolymeric DNA template-primers were determined by inhibition of DNA polymerase activity on a homopolymeric template-primer as described previously (31). Lyophilized heteropolymeric oligonucleotides were resuspended in 10 mM Tris-HCl pH 7.4 and 1 mM EDTA, and the concentrations determined from their UV absorbance at 260 nm. Template-primers were annealed by heating a solution of 5 IIM template (expressed as 3'-OH) with an equivalent concentration of primer to 90°C for 3 min, incubating the solution for an additional 30 min at 50-60°C, followed by slow cooling to room temperature. The sequences ofthe oligonucleotides used were: PI, 5'-CGAGCCATGGCCGCTAG-3'; P2, 5'-l1lIT1l1T1lGCGGTGCCAGG-3'; T, 5'-CCTGGCACCGC-

AAAAAATCFGCCTAGCGGCCATGGCTCG-3'. Dephosphorylated primers were labeled by T4 polynucleotide kinase as described (31). Enzyme activities were determined using a standard reaction mixture (50 p1) containing 50 mM Tris-HCl pH 7.4, 100 mM KCI, 5 mM MnCl2 or MgCl2 (Klenow fragment), 25 jM [a-32P]dTTP, the indicated concentration of poly(dA)-p(dT)20 (expressed as 3'-OH primer termini), and 1 ,uM of competitor heteropolymeric DNA. Nucleotide incorporation does not occur with the competitor substrate since the correct nucleotide to be incorporated (i.e., dGTP when PI is annealed to T, see above) is not included in the reaction mixture. Reactions were initiated by addition of polymerase, incubated at room temperature for 12 min and stopped by the addition of 20 ,ul of 0.5 M EDTA. Quenched reaction mixtures were spotted on DE8 1 filter disks and dTMP incorporation was determined as described above. The apparent dissociation constant, Kd,app, for the heteropolymeric duplex was calculated from:

Nucleic Acids Research, 1995, Vol. 23, No. 9 1599 Appazent WA

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