pyreneâDNA Adducts Inhibit the DNA Helicase ... - ACS Publications

Chem. Res. Toxicol. 1996, 9, 179-187

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Benzo[a]pyrene-DNA Adducts Inhibit the DNA Helicase Activity of the Bacteriophage T7 Gene 4 Protein Yongqi Yong† and Louis J. Romano* Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Received June 21, 1995X

The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome. In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP. Previous investigations have demonstrated that the translocation of the gene 4 protein along singlestranded DNA is blocked by the presence of benzo[a]pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts. In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo[a]pyrene-DNA adducts. The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA. Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo[a]pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA.

Introduction DNA replication requires at least partial and transient unwinding of the double helix to expose the genetic information carried by DNA. DNA helicases, which are responsible for this process, have been shown to be indispensable components of almost all known DNA replication systems. In bacteriophage T7, this role is fulfilled by the product of expression of the gene 4. The T7 gene 4 protein, apart from being a DNA helicase (1, 2), also provides the primase activity essential for the initiation of lagging strand DNA synthesis (3, 4) and, in addition, contains a single-stranded DNA-dependent deoxyribonucleoside 5′-triphosphatase activity (1, 5). In the presence of dTTP, the gene 4 protein has been shown to assemble into a hexameric complex (6) that has recently been observed by electron microscopy (7). Binding to single-stranded DNA (ssDNA)1 has been shown to trigger an additional conformational change within the gene 4 protein complex, which is necessary for hydrolysis of dTTP (8). Presumably, it is the energy released from this process that is used to propel the translocation of the gene 4 protein hexamer and to fuel its unwinding of double-stranded DNA (1). The effects of numerous DNA adducts on the replication of DNA have been well documented and include base substitution or frameshift mutations, large deletions, or chain termination (9). Recently, the impact of bulky chemical DNA adducts on the activity of DNA helicases has received increasing study because, according to the current models (10), DNA helicases function as the vanguards of the protein complexes responsible for DNA replication, and therefore they would first to encounter * To whom correspondence should be addressed. Tel: 313-577-2584; Fax: 313-577-8822; E-mail: [email protected]. † Present address, Department of Medicine, Bronx Municipal Hospital Center, Bronx, NY 10461. X Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: single-stranded DNA, ssDNA; benzo[a]pyrene, B[a]P; 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, BPDE; Tris-borate-EDTA, TBE; sodium dodecyl sulfate, SDS; cisdiamminedichloroplatinum, cisplatin.

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any potential DNA damage. In the light of this, it is logical to postulate that any lesions in DNA that can prevent the DNA helicase from unwinding doublestranded DNA might have a major impact on the overall DNA replication process. For example, it has been demonstrated that the DNA unwinding activities of the Rad 3 DNA helicase of Saccharomyces cerevisiae (11, 12), helicase II of Escherichia coli (11, 13, 14), the dda protein of phage T4 (14), calf thymus DNA helicase E (15), the SV40 large T antigen DNA helicase, human Hela cell DNA helicase (16), and the RecB and RecA proteins of E. coli (17) are all inhibited by various types of damage in DNA. One of the most important classes of chemical agents capable of forming bulky DNA adducts is polycyclic aromatic hydrocarbons, compounds derived from the incomplete pyrolysis of combustible materials (9). Benzo[a]pyrene (B[a]P) is an important member of this extensive family and was among the first carcinogens identified. In mammals, B[a]P is metabolized primarily by the mixed function oxidase system in conjunction with other related enzymes to its ultimate carcinogenic form, 7,8dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (9, 18, 19). The metabolite of this biotransformation process exits in two diastereomeric forms, each of which, in turn, has two enantiomers, giving rise to a total of four configurations: (+)-anti-BPDE, (-)anti-BPDE, (+)-syn-BPDE, and (-)-syn-BPDE (9, 20, 21). These electrophilic species are highly reactive toward DNA and are capable of forming covalent DNA adducts; the major ones formed both in vitro (18, 22) and in vivo (22, 23) result from the formation of a covalent linkage between the C-10 position of BPDE and the N2-exocyclic amino group of a guanosine residue. The mutagenicity and carcinogenicity of BPDE are generally attributed to the (+)-anti-BPDE (BPDE-I) while the other stereoisomers are not thought to be tumorigenic (24, 25). The knowledge about how B[a]P-DNA adducts affect the metabolism of DNA at the molecular level is fundamental to the understanding of how modification of DNA by this bulky polycyclic hydrocarbon molecule ultimately © 1996 American Chemical Society

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leads to tumor development. Previous studies have demonstrated that B[a]P-DNA adducts block the movement of various RNA polymerases (26-28) and DNA polymerases (29-32), causing termination of RNA and DNA synthesis. However, because proper functioning of almost all DNA polymerases requires the cooperative participation of other enzymes, including DNA helicases, it is important to learn the impact of B[a]P-DNA adducts on these enzymes. Previous studies aiming at understanding the effects of B[a]P-DNA adducts on the T7 gene 4 protein revealed that these adducts inhibited its dNTPase activity (33) that presumably blocked the unidirectional translocation and DNA-unwinding activities of the gene 4 protein, since both these two functions depend on the energy released from hydrolysis of dTTP (2, 5, 34). Furthermore, the gene 4 protein was likely to be sequestered at the sites of the B[a]P adducts when it encountered these lesions in DNA (33). In the present study, we have further characterized the impact of B[a]P-DNA adducts on the enzymatic activities of the gene 4 protein, by directly measuring how these adducts affect this enzyme in unwinding double-stranded DNA, and have explored the possible mechanism for the antihelicase action of these adducts.

Experimental Procedures Materials. (A) Enzymes. The T7 gene 4 protein was purified from extracts of E. coli strain 71.18 which harbored a plasmid overexpressing the wild type gene 4 protein. The expression of the cloned gene 4 was achieved by a T7 RNA polymerase/promoter system (35). The gene 4 protein was isolated as described (36). The last column in the purification scheme, namely, hydroxyapatite chromatography, allowed us to obtain preparations of the gene 4 protein with different ratios between the 56 kDa and the 63 kDa forms (37), ranging from a 1:1 to 300:1 enrichment in the 56 kDa protein (data not shown). Unless stated otherwise, all experiments described in this report use gene 4 protein preparations in which the ratio of 56 kDa form to 63 kDa form was 300:1. All protein preparations were greater than 99% pure as judged by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (38) and staining with silver. The bacteriophage T4 DNA ligase was purchased from New England Biolabs (Beverly, MA). (B) DNA and Oligonucleotides. Single-stranded circular phage M13mp9 DNA was prepared as described (39). All synthetic oligonucleotides were ordered from Midland Certified Inc. (Midland, TX). All concentrations of oligonucleotides are expressed in terms of the numbers of the oligonucleotide molecules. The concentration of ssM13 mp9 DNA is expressed in terms of nucleotide equivalents. (C) Reagents and Buffers. Deoxythymidine β,γ-methylenetriphosphate was ordered from Amersham. dTTP was ordered from Pharmacia (Piscataway, NJ). All radioactive nucleotides were from ICN Biomedicals (Irvine, CA). Gluteraldehyde (50% v/v water solution) was purchased from Aldrich (Milwaukee, WI). (+)-7β,8R-[ring-3H]Dihydroxy-9R,10R-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) was ordered from Chemsyn Science Laboratories (Lenexa, KS). Methods. (A) Modification of Oligonucleotides with B[a]P. Modification of oligonucleotides with (+)-BPDE-I was performed as described (40). After the incubation, the mixture of the modification reaction was extracted 10 times with watersaturated diethyl ether, which was followed by three consecutive ethanol precipitations to remove the excess BPDE-I. The number of adducts per oligonucleotide molecule was calculated from the ratio of 3H decays to the concentration of the oligonucleotide obtained from measuring the UV absorbance at 260 nm. (B) Preparation of Oligonucleotide Substrates for Helicase Assays. The substrate used in the initial helicase assays

Yong and Romano (Figure 1) consists of two oligonucleotides 50 bases and 60 bases long, respectively, annealed together to form a partial duplex. The 50 mer was 32P labeled at the 5′-end. The annealing reaction was carried out in 6 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 50 mM NaCl, and 1 mM dithiothreitol. The annealing mixture containing the 60 mer and the 32P-labeled 50 mer were incubated at 65 °C for 3 min, followed by cooling slowly to 30 °C over a period of 30 min. The mixture was then loaded into a 15% nondenaturing polyacrylamide gel cast in 1× Trisborate-EDTA (TBE) buffer. Electrophoresis was carried out at 250 V and 4 °C with 1× TBE as the running buffer. The radioactive bands were visualized by autoradiography, and the band corresponding to the annealed helicase substrate was cut from the gel. The gel slice was crushed, and the substrate molecules were eluted from the crushed gel by soaking in 300 mM sodium acetate (pH 5.2), 10 mM magnesium acetate, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS) at 25 °C for 5 h. The helicase substrate was then precipitated with ethanol, redissolved in a buffer consisting of 40 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM dithiothreitol, and stored at -20 °C. Structurally, this helicase substrate resembled a replication fork which contained a double-stranded region 38 base pairs in length, a 22 base long single-stranded tail with a 5′-end, and a 12 base long single-stranded tail bearing a 3′-end. To prepare a helicase substrate with a longer double-stranded region (Figure 4A), the 32P-labeled 50 mer was first annealed to the 60 mer as described above, after which two of the partial duplex molecules described above were ligated together via their blunt ends. The ligation reaction (50 µL) was carried out in 50 mM Tris-HCl (pH 7.6), 50 mM NaCl, 7 mM MgCl2, 10 mM dithiothreitol, 0.5 mM ATP, 50 µg of bovine serum albumin, 15% poly(ethylene glycol) 8000, and 150 units/mL T4 DNA ligase. The ligation mixture was incubated at 25 °C for 18 h, the products of the ligation reaction were then fractionated with a 15% nondenaturing polyacrylamide gel, and the blunt-end ligated partial duplex shown in Figure 4A was recovered from the gel as described above. The resulting construct consisted of two 110 base long strands, with a 76-base-pair long doublestranded region in the middle. The gel-purified ligation product was examined with polyacrylamide gel electrophoresis in the presence of 7 M urea. Typically, more than 98% of the strands were found to be completely ligated. (C) Helicase Assay. Helicase assays of the gene 4 protein were carried out in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, and 1 mM dTTP, at 30 °C for the indicated period of time. The reaction mixture (20 µL) contained typically 1.0 pmol of the partial duplex oligonucleotide substrate and the indicated amount of the gene 4 protein. The reaction was stopped by adding 10 µL of 100 mM EDTA (pH 8.0), 40% glycerol, 0.1% bromophenol blue, and 0.1% xylene cyanol. For time course experiments, the reaction was scaled up, aliquots were taken out at the times specified, and the reactions were stopped accordingly. The reaction products were fractionated with a 15% nondenaturing polyacrylamide gel cast in 1× TBE buffer. Electrophoresis was carried out at 15 V/cm and 4 °C, with 1× TBE as the running buffer. After the electrophoresis, the radioactive bands were visualized by autoradiography. To quantitate the helicase activity of the gene 4 protein, the radioactive bands corresponding to both the substrate and the displaced strand were cut from the gel and were counted in a liquid scintillation counter. Results were expressed as the percentage of total substrate displaced. In every case, the data presented in this paper are the average of at least four different assays. (D) Gel-Retardation Assay. The DNA-binding reaction mixtures (10 µL) were prepared in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM dithiothreitol. They typically contained 0.1 µM of the gene 4 helicase, 0.02 µM of a 32P-labeled oligonucleotide, and 2 mM thymidine triphosphate (dTTP). The optimal level of challenge DNA (250-fold) was determined by forming these complexes and adding increasing amounts of ssM13 DNA. At the end of the incubation, 1 µL of a diluted gluteraldehyde (glutaric dialdehyde) solution was then added

Benzo[a]pyrene-DNA Adducts Inhibit a DNA Helicase

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 181

Figure 1. Schematic representation of the helicase substrate. The helicase substrate was constructed by annealing the 50 mer and the 60 mer together as described under Experimental Procedures. The 50 mer was labeled at the 5′-end with [32P]ATP. The double-stranded region of this construct is 38 base pairs long, and the single-stranded 5′-tail and the 3′-tail were 22 and 12 bases in length, respectively. The sequences of the 60 mer and the 50 mer are also shown. to each reaction to a final concentration of 0.02%. The mixtures were incubated for a further 20 s, after which 6 µL of stop buffer (buffer B) was added into each tube and the mixtures were loaded immediately onto a 6% nondenaturing polyacrylamide gel cast in 0.25× TBE buffer. Electrophoresis was carried out at 12 V/cm and 4 °C with 0.25× TBE as the running buffer. The radioactive bands in the gel were visualized by autoradiography. To quantitate the DNA-binding activity of the gene 4 protein, the radioactive bands were cut from the gel and the radioactivity was measured in a liquid scintillation counter. For the reactions that were challenged with single-stranded DNA, the appropriate amount of single-stranded M13mp9 DNA was added into the DNA-binding reaction mixtures described above after 10 min of preincubation; the reaction mixtures were incubated for the additional periods of time as indicated and were then subjected to gluteraldehyde cross-linking and electrophoresis as described above.

Results Strand-Specific Effects of B[a]P-DNA Adducts on DNA Helicase Activity of Gene 4 Protein. The gene 4 protein is a multifunctional enzyme that contains DNA helicase, primase, and DNA-dependent dTTPase activities. One of the characteristics of the gene 4 protein is that it requires the presence of two single-stranded tails, one with a 5′-end and the other with a 3′-end, at the junction of the duplex in order to unwind doublestranded DNA (2). The minimal length for the 5′-tail is 12 bases, while that for the 3′-tail is 7 bases. The helicase substrate used in our reactions consisted of two oligonucleotides annealed to each other to form a partial duplex (Figure 1). The two oligonucleotides were 50 bases and 60 bases long, respectively. This construct contained a 38-base-pair double-stranded region, a 22 base long single-stranded tail with a 5′-end, and a 3′-tail 12 bases in length, a structure reminiscent of a replication fork. With this substrate, the gene 4 protein can bind to the 5′-single-stranded tail (part of the 60 mer), translocate from 5′ to 3′ to the junction of the fork, unwind the double-stranded region, and displace the other strand (the 50 mer). In the presence of dTTP, the gene 4 protein was able to separate about 35% of the unmodified helicase substrate molecules over a 20 min period of time when a slight molecular excess of the gene 4 protein was present (Figure 2A,B). However, modification of the 60 mer with B[a]P had a rather dramatic impact on the ability of the gene 4 protein to unwind the double-stranded region of the helicase substrate (Figure 2A,B). The presence of on

Figure 2. Strand-specific inhibition of the DNA helicase activity of the gene 4 protein by B[a]P-DNA adducts. (A) DNA helicase activity was measured as described under Experimental Procedures in reactions containing the gene 4 protein (62.5 nM) and the substrate (50 nM) shown in Figure 1. The left four lanes used a template containing no B[a]P adducts while the right four lanes used a template containing 1.3 B[a]P adducts in the 60 mer. (B) Unwinding was measured by cutting out the bands shown in panel A and counting in a scintillation counter. The open circles are the results obtained using an unmodified template, while the closed squares are 1.3 B[a]P adducts per template. An identical experiment was performed using a 60 mer containing 3.6 B[a]P adducts per template (closed triangles). (C) The DNA helicase reactions were carried out as described for panel B except that the B[a]P adducts were located in the 50 mer (the displaced strand). The helicase substrates used in these reactions were either unmodified (open circles) or B[a]Pmodified (closed circles) containing 3.7 B[a]P adducts per 50 mer.

average 1.3 B[a]P adducts per 60 mer molecule was able to suppress more than 50% of the DNA helicase activity of the gene 4 protein in comparison with the unmodified substrate. More extensive modification of the 60 mer to a level of 3.6 B[a]P adducts per molecule resulted in even greater inhibition (Figure 2B). These results indicated that the presence of B[a]P-DNA adducts inhibited the DNA helicase activity of the gene 4 protein in a dosedependent manner. Note that the 60 mer was the strand on which the gene 4 protein bound and translocated with a 5′ to 3′ polarity during its helicase action and that all of the B[a]P adducts present in the helicase substrate were located within the double-stranded region. This latter fact showed that the observed inhibition was not a consequence of preventing the gene 4 protein from


Yong and Romano

Figure 3. Effect of ionic strength on the DNA helicase activity of the gene 4 protein. (A) The B[a]P adducts were located in the 60 mer. The helicase reactions of the gene 4 protein were carried out at different NaCl concentrations as indicated. Otherwise, the reaction conditions were identical to Figure 2. The helicase substrates either were unmodified (open circles) or contained 1.3 (closed squares) and 3.6 (closed triangles) per oligonucleotide molecule, respectively. (B) The B[a]P adducts were located in the 50 mer (the displaced strand). The conditions of the helicase reactions were identical to panel A. The helicase substrates either were unmodified (open circles) or contained 3.7 B[a]P adducts per 50 mer (closed circles).

binding to the single-stranded region in the helicase substrate, but rather was a direct result of blocking the DNA-unwinding process of the gene 4 protein. To test whether the effects of B[a]P on the gene 4 protein as observed above were strand-specific, the 50 mer modified to an extent of 3.7 B[a]P adducts per molecule was annealed to the unmodified 60 mer to form the partial duplex substrate. The 50 mer provided the 3′-single-stranded tail for the substrate construct, and due to the 5′ to 3′ directionality of the translocation of the gene 4 protein, binding of the gene 4 protein to this strand cannot result in the unwinding of the duplex. When the gene 4 protein was tested for its helicase activity with this substrate, the extent to which it was unwound and the kinetics of the unwinding reaction were almost indistinguishable from those for the unmodified substrate (Figure 2C). This showed that the DNA helicase activity of the gene 4 protein was inhibited only when it encountered a B[a]P-DNA adduct in the strand on which it translocates during the unwinding of doublestranded DNA. Effects of Salt Concentrations on DNA Helicase Activity of Gene 4 Protein. Even though DNA unwinding by the gene 4 protein was markedly inhibited by the presence of B[a]P adducts on the duplex region of the lagging strand of the replication fork, a substantial portion of the helicase activity still remained. It is possible that the residual activity resulted from either a slow bypass of the adducts or dissociation of the gene 4 protein followed by reassociation past the adduct site. Alternatively, it is possible that the B[a]P-DNA adducts completely inhibited the helicase activity of the gene 4 protein, but that the double-stranded region that remained was too short to maintain the duplex. The latter explanation seemed more likely since B[a]P adducts were shown to completely abolish the DNA-dependent dTTPase activity of the gene 4 protein (33). To distinguish these possibilities, we tested the effects of increasing the ionic strength of the medium on the DNA helicase activity of the gene 4 protein using unmodified or B[a]P-modified helicase substrates. Because higher concentrations of NaCl should stabilize the double-helical structure of DNA, we reasoned that the effect of increasing NaCl concentrations on unwinding should be greatest for the modified template. Thus, if

the gene 4 protein were indeed completely inactivated after it encountered a B[a]P-DNA adduct, the remaining short stretch of duplex downstream of the adduct would be stabilized by higher salt concentrations, and therefore, one would expect that with the modified substrate strand displacement would be more inhibited at higher salt concentrations. With the unmodified substrate, the strand displacement would be less affected by higher salt concentration since the gene 4 protein could still unwind the unmodified substrate completely under such conditions. Using the level of helicase activity obtained in the standard buffer used in the helicase reactions (50 mM NaCl) as 100% activity for each of the templates, we tested the DNA helicase activity of the gene 4 protein at 100, 150, and 200 mM NaCl with both the unmodified and modified helicase substrates (Figure 3). With the unmodified partial duplex as the substrate, the DNA helicase activity of the gene 4 protein remained unchanged at up to 150 mM NaCl, and only at a concentration of 200 mM NaCl did the helicase activity of the gene 4 protein drop to about 50% of the original level. With the BPDE-modified substrate where the B[a]P adducts were in the 60 mer (Figure 3A), the percent displacement of the substrate dropped rapidly from its original level as the salt concentration increased, and at 200 mM NaCl with the most highly modified substrates the helicase activity was almost undetectable (Figure 3A). When the adducts were located in the 50 mer (displaced strand), the effect of salt on unwinding was the same as if both strands were unmodified (Figure 3B). These results are consistent with the hypothesis that the gene 4 protein was inactivated by the B[a]P-DNA adducts and that the residual strand displacement seen in Figure 2A was actually due to spontaneous melting of what remained of the double-stranded region downstream of the arrested gene 4 protein, and they lend further support to the hypothesis that the inhibition of the DNA helicase activity of the gene 4 protein by B[a]P-DNA adducts was strand-specific. DNA Helicase Activity of Gene 4 Protein Is Completely Blocked by B[a]P-DNA Adducts. The results of the experiments described above suggested that the double-stranded regions of the helicase substrate used in these experiments were too short to show the full



Figure 4. B[a]P-DNA adducts completely inhibits the helicase activity of the gene 4 protein. (A) Schematic representation of the blunt-end ligated helicase substrate. The partial duplex described in Figure 1 was labeled at the 5′-end of the 50 mer with [γ-32P]ATP. Ligation and purification of the blunt-end ligated substrate were carried out as described under Experimental Procedures. The double-stranded region of this construct consisted of 76 base pairs. The single-stranded 5′- and 3′-tails were 22 and 12 bases long, respectively. For the B[a]P-modified substrate, there were on average 1.3 adducts in the 60 mer portion of each strand. (B) DNA helicase reactions with the unmodified substrate. The DNA helicase reactions and the separation of the reaction products were carried out as described. The compositions of these reactions and their reaction time were indicated. Lane A: A heat-denatured sample of the helicase substrate. Lane B: The helicase reaction was carried out in the absence of dTTP. The positions of the helicase substrate and the labeled displaced strand were indicated. (C) DNA helicase reactions with the B[a]P-modified substrate. The DNA helicase reactions were carried out as described in panel B. The B[a]P-modified substrate was used. (D) Quantitation of strand displacement in the reactions shown in panels B and C. Open circles: Unmodified helicase substrate. Closed circles: B[a]P-modified substrate.

effect of the B[a]P-DNA adducts on the helicase activity of the gene 4 protein. To prepare a partial duplex with a substantially longer double-stranded region, we carried a ligation reaction with the monomer shown in Figure 1 to form a dimer shown in Figure 4A. This product has a symmetrical structure having a double-stranded region 76 bases in length and adducts located in the 60 mer regions, ensuring that the gene 4 protein would encounter an adduct regardless of the strand of entry. As with the prior experiments, the enzyme/DNA ratio was 1.25:1 so that the chance of the same substrate molecule being attacked simultaneously by two gene 4 protein complexes from two sides was minimal. Using this substrate, the unwinding by the gene 4 protein was almost completely abolished even when there were on average only 1.3 B[a]P adducts in each strand (Figure 4C,D), suggesting that rather than simply slowing down the progression of the gene 4 protein, the B[a]P-DNA adducts were able to completely block the helicase activity. Sequestration of Gene 4 Protein on B[a]P-Damaged Single-Stranded DNA. Previous experiments suggested that the gene 4 protein was sequestered at the sites of the B[a]P adducts (33). To gain a further insight into the interaction between the gene 4 protein and B[a]P-modified single-stranded DNA, the gene 4 protein was first incubated with an excess of either the unmodified or B[a]P-modified oligonucleotide for 10 min in the presence of dTTP, enabling the binding reactions to attain equilibrium, and then the unmodified helicase substrate shown in Figure 1 was added into the reaction mixtures. The incubation was continued for an ad-

ditional 10 min to allow the gene 4 protein not trapped by the single-stranded competing oligonucleotides to unwind the substrate, after which the reactions were stopped and the products were fractionated with nondenaturing polyacrylamide gels. If the gene 4 protein was indeed sequestered by the B[a]P-damaged single-stranded DNA after it encountered the adducts, one would expect that there would be less of this enzyme available in the reaction mixture to unwind the helicase substrate. In essence, the helicase reaction served as a measure of the relative amount of the gene 4 protein molecules not occupied by the 50 mer. As a control, we first tested the relationship between the concentration of the gene 4 protein and the efficiency of strand displacement (Figure 5A). The percent of the helicase substrates unwound over a 10 min period was directly proportional to the concentration of the gene 4 protein in the reaction mixture. This linear relationship was maintained until the concentration of the gene 4 protein reached 250 nM (Figure 5A). Therefore, the concentration of the gene 4 protein chosen for the subsequent experiments was 150 nM, well within the linear range. When the gene 4 protein was preincubated with the B[a]P-modified 50 mer, we observed substantially more reduction in the helicase activity of the gene 4 protein than when it was preincubated with the same amount of the unmodified 50 mer (Figure 5B,C). Presumably, this difference reflects the smaller amount of the gene 4 protein available for unwinding the helicase substrate.


Figure 5. Effect of B[a]P adducts on the stability of the gene 4 protein-DNA complex. (A) Effects of the concentration of the gene 4 protein on the efficiency of its helicase reaction. The DNA helicase reactions which contained the helicase substrate described in Figure 1, the separation of the reaction products, and the quantitation of the strand displacement were carried out as described under Experimental Procedures. The percentage of the total substrate unwound was plotted against the concentration of the gene 4 protein used. (B) The gene 4 protein (150 nM) was preincubated with the indicated amount of either the unmodified or the B[a]P-modified 60 mer at 30 °C for 10 min, after which the unmodified helicase substrate described in Figure 1 was added to a final concentration of 50 nM; the reactions were incubated for another 10 min, stopped, and analyzed by electrophoresis under nondenaturing conditions as described. The B[a]P-modified 60 mer contained 1.3 B[a]P adducts per molecule. (C) Quantitation of the strand displacement in the reactions in panel B was carried out as described. The helicase activity was expressed as the percentage of the activity observed for a control reaction where the gene 4 protein was preincubated in the absence of the competing 60 mer.

Gene 4 Protein Forms a Stable Complex with B[a]P-Modified Single-Stranded DNA. To directly observe the gene 4 protein-DNA complex, we performed gel-retardation experiments with the gene 4 protein and B[a]P and unmodified oligonucleotides. Prior studies have shown that the functionally active form of the gene 4 protein was a hexamer and that the preassembled hexamer could be stabilized by cross-linking with gluteraldehyde, a chemical agent which forms covalent cross-linkages between protein molecules.2 Cross-linking with gluteraldehyde stabilized the quaternary structure 2

Yong and Romano, unpublished results.

Yong and Romano

of the active gene 4 protein complexes, allowing these complexes to remain intact and associated with singlestranded DNA upon electrophoresis under nondenaturing conditions, so that the protein-DNA complexes formed can be directly analyzed by gel-retardation experiments. The chemical cross-linking reaction lasts for only 20 s following a 10 min incubation of the gene 4 protein and single-stranded DNA probe, and therefore it was not likely to cause a perturbation of the equilibrium of the DNA-binding reaction of the gene 4 protein. To evaluate the stability of the protein-DNA complexes formed by the gene 4 protein and the unmodified or B[a]P-modified single-stranded DNA, we first incubated the gene 4 protein with excess 32P-labeled oligonucleotide (the 50 mer) to form protein-oligonucleotide complexes, and then these preformed complexes were challenged with the addition of a large excess of singlestranded M13mp9 DNA. Because any hexameric gene 4 protein complexes that dissociate from the 50 mer probe would be trapped by the ssM13 DNA, the dissociation of the protein-50 mer complexes can be directly monitored by measuring the remaining gene 4 protein-50 mer complex. Time courses of the level of complex remaining indicate that the gene 4 protein complex was substantially more resistant to the challenge than those formed by the gene 4 protein and the unmodified 50 mer (Figure 6A,B). The level of complex was quantified by determining the radioactivity in each band, and these values are shown in Figure 6C. It is clear from the differences in the stability of these complexes that the dissociation of gene 4 protein-DNA complex is significantly slower when the DNA contains B[a]P adducts. Analysis of the data shown in Figure 6C suggests that the complex to the B[a]P-modified DNA is a two population phenomenon with a relatively fast dissociation occurring at a rate similar to that which occurs on unmodified DNA followed by a very slow dissociation that decreases little from 2 to 15 min. In fact, we have measured this dissociation at extended times and find that, after 2, 4, and 6 h, 50%, 41%, and 19%, respectively, of the complex remains. One possible explanation for this bimodal curve is that the fast component represents binding of the gene 4 protein to the modified 50 mer on the 3′-side of the adduct while the slow component represents binding to the 5′-side of the adduct. In the former case, 5′ to 3′ translocation by the gene 4 protein would not be blocked by the B[a]P adducts, resulting in a complex resembling that occurring on unmodified DNA, while in the latter translocation would bring the gene 4 protein to the adduct site where it presumably becomes strongly bound. Finally, although the complex formed between the gene 4 protein and B[a]P-modified single-stranded DNA is very stable, it could be completely disrupted by simply heating at 100 °C for 1 min (data not shown). This suggests that the complex is not likely to be the result of the formation of covalent linkage between the gene 4 protein and the B[a]P-DNA adducts.

Discussion DNA polymerases require the assistance of a variety accessory proteins during DNA replication. These enzymes, which normally include DNA helicases, are thought to participate in DNA synthesis as components of a functionally integrated replication complex or replisome (41). The role of the DNA helicase is to unwind



Figure 6. The gene 4 protein forms a more stable complex with the B[a]P-modified DNA. The DNA-binding reactions of the gene 4 protein were carried out as described under Experimental Procedures, with either the unmodified or B[a]P-modified 50 mer as the probe for binding. (A) The protein-DNA complex formed by the gene 4 protein and the unmodified 50 mer was challenged with single-stranded M13 DNA. The compositions of the DNA-binding reactions (80 µL) were indicated. The reaction were incubated at 30 °C for 10 min, after which it was challenged with a 250-fold excess of unlabeled single-stranded M13mp9 DNA. The reaction was allowed to continue at 30 °C and 10 µL aliquots were taken from the reaction mixture at the specified time points; these were cross-linked with gluteraldehyde and analyzed by electrophoresis as described under Experimental Procedures. Lane A: A control binding reaction where the gene 4 protein was incubated with the 50 mer probe in the absence of the dTTP. (B) The protein-DNA complexes formed by the gene 4 protein and the B[a]P-modified 50 mer were challenged with single-stranded M13 DNA. The reactions and analysis were carried out as described for panel A. The modified 50 mer contained 3.7 B[a]P adducts per molecule. (C) Quantitation of the results of the reactions in panels A and B. The percentages of the total 50 mer probe bound by the gene 4 protein were determined. These were expressed as the percentages of the values observed for the reactions not challenged with single-stranded M13 DNA (the 0 min reactions in panels A and B). Open circles: Unmodified 50 mer; closed circles: the B[a]P-modified 50 mer.

the DNA duplex ahead of the advancing DNA polymerase to expose the template strands for DNA synthesis. Therefore, this component of the replisome is likely to be the first to encounter any lesions in the DNA during DNA replication. In the bacteriophage T7 replication system, the DNA helicase activity is provided by the gene 4 protein (1, 2). In the present study, we have investigated the impact of B[a]P-DNA adducts on the DNA helicase and singlestranded DNA-binding activities of the gene 4 protein. Our results indicated that the gene 4 protein encountered great difficulty in unwinding a duplex damaged by B[a]P. In addition, the effect of B[a]P-DNA adducts on the DNA helicase activity of the gene 4 protein was strand-specific; a B[a]P adduct prevents the gene 4 protein from unwinding the duplex only when it was located in the strand on which the gene 4 protein is presumably bound and translocating. When the adduct is located in the opposite strand, the DNA helicase activity of the gene 4 protein is not affected to any appreciable extent. The DNA adducts formed by other chemical agents also displayed similar strand specificity. For example, cyclobutane pyrimidine dimers (11) and cisplatin-GG dimers and CC-1065-DNA adducts (12) inhibit the S. cerevisiae Rad 3 protein only when they are located on the same strand that the Rad 3 protein binds and translocates. However, E. coli helicase II, an enzyme which translocates along single-stranded DNA with a 3′ to 5′ polarity, is only inhibited by CC-1065-DNA adducts situated in the displaced strand (42). It is interesting that the CC-1065-DNA adduct covers a 3- and a 1-basepair region to the 5′- and 3′- sides of the covalently modified adenine, respectively (43), and has its major helix-winding and helix-stiffening effects to the 5′-side of this adenine residue. It was proposed that the CC1065 adduct inhibits a DNA helicase when the helixwinding effect of the adduct is transmitted in the opposite direction to the helicase unwinding activity (42). Whether this mechanism is related to the effects we observe for the B[a]P-DNA adducts is not known. It is interesting to attempt to understand how the strand specificity of the inhibition of the B[a]P-DNA

adducts relates to the mechanism of the DNA unwinding by the gene 4 protein. Previous studies have suggested that the functionally active form of the gene 4 protein is a hexamer (6, 7) and that the hexamer interacts with both strands of DNA simultaneously.2 Since the gene 4 protein unwinds double-stranded DNA unidirectionally, one would expect that the contacts made by the gene 4 protein hexamer with each individual strand may not be equivalent. Our observation that the B[a]P adducts inhibit the progression of the gene 4 protein only when they are situated in the strand where this protein presumably binds and translocates clearly indicated that the contacts made by the gene 4 protein with the two strands of a DNA duplex are not symmetric: one of the strands appears to bear the brunt of the interaction, while the contacts made with the other strand serve a secondary yet presumably indispensable role. The observation that the gene 4 protein is more resistant to the B[a]P adducts located in the displaced strand of the replication fork supports the hypothesis that the progression of the gene 4 protein complex during DNA unwinding is initiated through its stronger interaction with this strand. The gene 4 protein is sequestered on single-stranded DNA damaged by B[a]P-DNA adducts, forming a highly stable protein-DNA complex. The trapped gene 4 protein hexamer is likely to be situated in close proximity to the B[a]P adduct itself, since it has been demonstrated that the segment of DNA near the site of the B[a]P adduct is specifically protected from nuclease digestion by the gene 4 protein (33). The same phenomenon has also been observed for other DNA helicases with DNA adducts derived from a variety of chemical agents. Among these, the S. cerevisiae Rad 3 protein was found to be sequestered by single-stranded DNA containing either cyclobutane pyrimidine dimers, cisplatin adducts, or CC-1065 adducts (12). The trapping of the Rad 3 protein by these DNA lesions has been implicated to be fundamental to the potential roles that this protein plays in yeast nucleotide excision repair pathway, i.e., to search and locate DNA damages and to recruit other DNA repair enzymes to the site of these lesions. In the case of the


T7 gene 4 protein, the biological significance of the trapping of the gene 4 protein at the site of an adduct is not clear at this juncture; however, one of the ramifications of these results is the potential enhancement of the adduct blocking effect of the replication complex at the adduct site. At the present time, it is not completely understood how the gene 4 protein is sequestered by B[a]P-DNA adducts. Previous studies have suggested that the presence of B[a]P adducts in single-stranded DNA severely inhibits the DNA-dependent dTTPase activity of the gene 4 protein, suggesting that when the gene 4 protein encounters a bulky adduct, the conformational changes necessary for its hydrolysis of dTTP are disrupted because of the spatial constraints imposed by the close contact made by this protein with the bulky adduct (33). Recently, we have used gel-retardation experiments to demonstrate that the binding affinity of the gene 4 protein to single-stranded DNA is greatly influenced by the type of nucleotide cofactor to which it is associated: in the presence of dTDP the binding affinity is at least 1 order of magnitude lower than that when it is coupled with dTTP (8). Thus it is possible that the inability of the gene 4 protein to hydrolyze dTTP after it encounters a bulky adduct, which leads to its continuous association with this nucleotide cofactor (33), results in the gene 4 protein forming a much more stable complex with the B[a]P-damaged single-stranded DNA. Future studies should not only determine the mechanism of the antihelicase activity of these adducts, but may provide important clues to understand how DNA helicases unwind double-stranded DNA.

Acknowledgment. This investigation was supported by Public Health Service Grants CA35451 and CA40605 awarded by the National Cancer Institute, Department of Health and Human Services.

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