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Originally published In Press as doi:10.1074/jbc.M510122200 on December 27, 2005

J. Biol. Chem., Vol. 281, Issue 9, 6000-6009, March 3, 2006
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Inhibition of Werner Syndrome Helicase Activity by Benzo[a]pyrene Diol Epoxide Adducts Can Be Overcome by Replication Protein A*Formula

Saba Choudhary{ddagger}1, Kevin M. Doherty{ddagger}1, Christopher J. Handy§, Jane M. Sayer§, Haruhiko Yagi§, Donald M. Jerina§, and Robert M. Brosh, Jr.§2

From the {ddagger}Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland 21224 and the §Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892

Received for publication, September 14, 2005 , and in revised form, December 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N6 of deoxyadenosine (dA) or N2 of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA damage evokes a cellular response by a genome surveillance system that senses DNA structural perturbation at the site of the lesion and elicits an appropriate response that may involve direct repair of the lesion, stabilization of the replication fork, or induction of apoptosis (1). Cellular pathways of DNA metabolism are influenced by DNA lesions, and since DNA unwinding enzymes known as helicases are among the first proteins to encounter DNA damage, it is of interest to understand how helicase action is modulated by interaction with chemically modified DNA. Although the mechanism for DNA unwinding has been studied for several helicases (2–4), only limited information is available regarding how specific covalently linked adducts affect helicase function. Of particular interest to us has been the Werner (WRN)3 helicase that is defective in the premature aging and genome instability disorder Werner syndrome (WS) (5). The WRN helicase belongs to the RecQ family of Superfamily 2 DNA helicases (5) and has been shown to unwind double-stranded DNA in a 3' to 5' direction with respect to the strand on which it is presumed to translocate (6). Biochemical characterization of the DNA substrate specificity of WRN helicase has revealed that the enzyme has the ability to target DNA replication/repair intermediate structures by its ability to recognize DNA junctions (7). Analysis of presteady state kinetics of WRN helicase indicated that the burst amplitude (unwound 19-base pair (bp) forked duplex molecules) was very close to the WRN protein concentration, suggesting that WRN can function as a monomer to unwind the duplex DNA substrate (8). WRN is a much more processive helicase in the presence of the human single-stranded DNA binding protein replication protein A (RPA), enabling the enzyme to unwind DNA duplexes as long as ~850 bp (6, 9). The biological significance of the WRN-RPA interaction is not well understood but is likely to relate to the implicated role of RPA in DNA replication, repair, or recombination.

The replication (10–13) and recombination (14–16) defects observed in WS cells may reflect abnormal processing of specific structures associated with the replication fork or a DNA recombination intermediate. WRN and other RecQ helicases have been proposed to function in repairing replication forks that have been stalled by DNA damage (17–19). In addition, RecQ helicases may play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur (20). Studying the effects of DNA damage on the activity of RecQ helicases may lead to insight on how these helicases act upon encountering specific DNA adducts and how the absence of a given RecQ helicase contributes to genomic instability.

Benzo[a]pyrene (BaP) in the diet and in the air from combustion of fuel and tobacco is one of the most potent carcinogens to which humans are frequently exposed (21). A metabolic pathway involving cytochrome P450 and epoxide hydrolase converts BaP to BaP diol epoxides (BaP DEs) (22). Two diastereomers (each of which exists as a pair of enantiomers) of a given bay region DE are metabolically possible, one diastereomer (DE-1) in which the benzylic hydroxyl group and the epoxide oxygen are cis and one in which these two groups are trans (DE-2). Cis or trans opening of the epoxide ring of BaP DE by the exocyclic amino group N6 of adenine or N2 of guanine results in covalent DNA adducts (Scheme 1) (23). These bulky polycyclic aromatic hydrocarbon adducts impede DNA replication and induce mutations by causing replication errors. In the present study, we utilize only adducts derived from the DE-2 diastereomer.


Figure 1
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  		SCHEME 1.
Structures of the optically active BaP DEs and their purine nucleoside adducts, where B represents a dG or dA moiety attached via its exocyclic amino group, N2 or N6, respectively, as shown. The partially saturated benzo-ring that is the site of covalent attachment of the nucleoside is shown in boldface type. The absolute configuration at C-10 is retained upon cis opening and inverted upon trans opening of each DE.

 
The stereoisomeric BaP DE adducts at dG and dA provide a variety of structural motifs within duplex DNA that have been well characterized by NMR studies (24, 25). For cis-opened BaP DE-dG adducts, both 10R and 10S enantiomers cause flipping out of both the modified base and its complement from the DNA helix (26, 27). BaP DE-dG trans adducts occupy the minor groove, with the hydrocarbon moiety of the 10R and 10S adducts oriented in opposite directions relative to the helix axis (toward the 3' and 5' ends of the modified strand, respectively) and do not significantly distort the double helix of B-form DNA (28–30). In contrast, both cis (31) and trans (32–35) opened dA adducts intercalate between base pairs such that the aromatic moiety of the 10R adducts inserts on the 5' side of the adducted base, whereas the aromatic moiety of the 10S adducts inserts on the 3' side. Based on the NMR structures, intercalation of the hydrocarbon results in buckling and twisting of the base pairs in the immediate vicinity of the adduct as well as local unwinding and overall bending of the helix axis.

The BaP DE DNA adducts are partially resistant to cellular repair processes (36–39) and thus are likely to be encountered by other DNA-processing enzymes. Although DNA polymerases are potential targets (40–46), the ability of polycyclic aromatic hydrocarbon-DE DNA adducts to interact adversely with other enzymes such as topoisomerases (47, 48) may also contribute to their carcinogenic effects. In addition to these classes of DNA-metabolizing enzymes, helicases are likely to be subject to the adverse effects of DNA lesions induced by environmental chemicals.

In the current study, we observe that the effects of BaP DE adducts with defined structural motifs on WRN helicase activity are specific to the nature of the modified base as well as to the strand on which the lesion is located. Thus, a single BaP DE-dG adduct with either cis or trans, R or S stereochemistry inhibited WRN in a strand-specific manner, whereas either a cis or trans, R or S BaP DE-dA adduct had little effect. These results suggest that the effects of BaP DE adducts on WRN helicase activity are related to the base to which the adduct is covalently attached and dependent on the structure of the BaP DE DNA adduct, such that WRN helicase activity is sensitive to BaP DE dG adducts, which cause flipped out bases or occupy the minor groove. The single-stranded DNA-binding protein RPA, which serves as an auxiliary factor for the WRN helicase, enabled WRN to overcome the inhibition exerted by either the cis or trans BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent DNA adducts that otherwise block WRN helicase acting alone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins—Recombinant hexahistidine-tagged WRN protein was overexpressed using a baculovirus/Sf9 insect system and purified as described previously (19). Recombinant RECQ1 helicase was overexpressed in insect cells using a baculovirus encoding recombinant human RECQ1 kindly provided by Dr. Alessandro Vindigni (International Centre for Genetic Engineering and Biotechnology) and purified as previously described (49). Purified recombinant UvrD helicase was kindly provided by Dr. Steven Matson (University of North Carolina at Chapel Hill). Purified human RPA containing all three subunits (RPA70, RPA32, and RPA14) was graciously provided by Dr. Mark Kenny (Albert Einstein Cancer Center).

Nucleotides, Oligonucleotides, and DNA Substrates—[{gamma}-32P]ATP was from PerkinElmer Life Sciences. Unadducted oligonucleotides were purchased from Lofstrand Technologies. Oligonucleotides containing diastereomerically pure cis- and trans-opened BaP DE-dA or BaP DE-dG adducts were synthesized using a semiautomated procedure, essentially as described (50), with a manual step for coupling of the BaP DE-dA (51, 52) or BaP DE-dG phosphoramidites (53) as their pure 10R or 10S diasteromers. The synthesized oligonucleotides were purified by reverse-phase high pressure liquid chromatography after removal of the 5'-protecting dimethoxytrityl group. Typically, each oligonucleotide was chromatographed twice, utilizing two different columns successively: 1) a Hamilton PRP-1 column (7 µm, 10 x 250 mm) at 25 °C (retention times between 16 and 18 min for all oligonucleotides) and 2) a Waters XTerra MS C18 column (2.5 µm, 10 x 50 mm) at 25 or 45 °C (retention times between 10 and 13 min at 45 °C). Elution was at 3 ml/min with a gradient that increased the proportion of solvent B in A from 0 to 35% over 20 min, where solvent A is 0.1 M (NH4)2CO3 buffer and B is a 1:1 mixture of A with acetonitrile, both adjusted to pH 7.0–7.5. The absolute configurations of the adducts are known from the diastereomerically pure reactant phosphoramidites (51–53). Single-stranded oligonucleotides with BaP DE-dA or BaP DE-dG adducts are shown in Table 1. The corresponding forked duplex substrates, prepared as previously described (7), are shown in Fig. 1, A and B.


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  		TABLE 1
Oligonucleotide sequences for DNA substrates with BaP DE-dA or BaP DE-dG adducts The adducted nucleotide is in boldface type.

 
Helicase Assays—Helicase assay reaction mixtures (20 µl) contained 30 mM HEPES (pH 7.4), 5% glycerol, 40 mM KCl, 100 ng/µl bovine serum albumin, 2 mM MgCl2, 2 mM ATP, 0.5 nM DNA substrate, and the indicated concentrations of WRN, RECQ1, or UvrD. Reactions were initiated with the addition of the indicated helicase and then incubated at 37 °C for 15 min. Reactions were quenched with 20 µl of Stop buffer (35 mM EDTA, 0.6% SDS, 25% glycerol, 0.04% bromphenol blue, 0.04% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide of the same sequence as the labeled strand of the DNA fork substrate. The products of the helicase reactions were resolved on nondenaturing 12% polyacrylamide gels. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager and quantitated using ImageQuant software (Amersham Biosciences). The percentage of helicase substrate unwound was calculated by the formula, percentage of unwinding = 100 x (P/(S + P)), where P is the product and S is the residual substrate. The values of P and S have been corrected after subtracting background values in controls having no enzyme and heat-denatured substrate, respectively. Helicase data represent the mean of at least three independent experiments, with mean ± S.D. shown by error bars.


Figure 2
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  		FIGURE 1.
Forked duplex DNA substrates with BaP DE adducts. A, solid and open triangles designate the hydrocarbon portions of the dA adducts from the trans and cis opening of the epoxide ring, respectively. The star indicates a 5'-32P label. The direction in which the symbols point indicates the orientation of the intercalated hydrocarbon relative to the modified adenine base: on the 3' side for S adducts and on the 5' side for R adducts. B, for the trans dG adducts, the solid triangles point in the direction in which the aromatic hydrocarbon portion of the adduct orients in the minor groove. For the cis dG adducts (rectangles), the hydrocarbon is intercalated with base displacement, and thus its orientation relative to the directionality of the DNA strand is not defined. Note that the adducted guanine base (not shown) to which the hydrocarbon is bonded orients toward the 5' end of its strand for both cis-R and cis-S dG adducts but is displaced into the minor groove for the R and into the major groove for the S adduct.

 
For helicase sequestration studies, WRN (3 nM in a 20-µl reaction) was preincubated with 0–500 fmol of the indicated unlabeled single-stranded DNA or unlabeled forked duplex DNA molecules in standard helicase reaction conditions as described above for 3 min at 37 °C. After the 3-min incubation, 10 fmol of radiolabeled forked duplex tracker substrate was added to the reaction mixture and incubated for 7 min at 37 °C. Reactions were quenched and resolved on native polyacrylamide gels as described above. Typically, 75–90% of the tracker helicase substrate was unwound in reactions lacking the competitor DNA molecule.

For kinetic helicase assays, 160-µl reaction mixtures containing the standard helicase reaction salts (see above), 0.5 nM duplex DNA substrate, 4.6 nM WRN in the presence or absence of 12 nM RPA were incubated for 0–4 min at 37 °C. 20-µl reaction volumes were removed and quenched as described above at the specified times. To correct for duplex destabilization by RPA, the background value for displaced strand (≤10%) in RPA alone reaction mixtures was subtracted from those values obtained for the WRN-RPA helicase reaction products. UvrD kinetic assays were conducted similar to the WRN kinetic assays except that UvrD (12 nM) was incubated with the substrate for 0–8 min at 37 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the effects of DNA structural perturbations induced by adducts formed from DEs of the chemical carcinogen BaP on WRN helicase activity, we have tested a series of related forked duplex DNA substrates with a covalently bonded site-specific BaP DE adduct positioned centrally in the 22-bp duplex region on one of the two DNA strands. The sequence chosen for placement of the adduct within the duplex is found in the coding sequence (exon 7) of the p53 gene (54). The site of the adducted base, BaP DE-dG or BaP DE-dA (Table 1), corresponded to p53 hotspot codons 248 and 249, respectively. The design of the DNA substrates used in this study enabled us to assess the effects of adduct stereochemistry, orientation, strand occupation, and nature of the DNA structural perturbation on WRN helicase activity.

For both the adducted (BaP DE-dA or BaP DE-dG) and unadducted forked duplex (22-bp) substrates, the intact oligonucleotide was released with minimal degradation (<2%) from the blunt end by WRN exonuclease as observed on native gels and confirmed on urea-denaturing gels (data not shown). Similar unwinding of the DNA substrates by an exonuclease-defective mutant WRN protein (WRN-E84A) was also observed (data not shown). These results are consistent with the previous observation that displacement of short (16- and 22-bp) duplex substrates by WRN helicase activity is more rapid than digestion by the WRN exonuclease activity (55).

Effect of BaP DE-dA Adducts on WRN Helicase Activity—We first tested substrates with a single BaP DE-dA adduct situated on the strand opposite to that on which WRN translocates (substrates 1–5; Fig. 1A) (Fig. 2A). For all of the five substrates tested, the percentage of duplex unwound depended on the concentration of WRN present in the reaction (Fig. 2A). The quantitative results from these assays demonstrated that WRN unwinding was not affected by the BaP DE-dA adducts residing in the nontranslocating strand, regardless of their stereochemistry.

We next tested substrates with a single BaP DE-dA adduct situated on the strand on which WRN translocates (substrates 6–10; Fig. 1A) (Fig. 2B). As before, the percentage of duplex unwound depended on the concentration of WRN present in the reaction (Fig. 2B). At the two highest concentrations of WRN tested, 2.3 and 4.6 nM, a statistically significant difference in WRN unwinding could be detected for certain substrates of this group. At 2.3 nM WRN, unwinding of the cis-R BaP DE-dA was reduced by ~1.5-fold compared with the unadducted substrate. At 4.6 nM WRN, unwinding of the cis-S and cis-R BaP-dA adducted substrates was reduced by ~1.7-fold. The trans S isomer showed slight inhibition at the highest concentration of WRN tested, although not as profound as the inhibition by the cis isomer. The trans-R configuration showed no inhibition at any level of WRN.

The possibility exists that when WRN helicase encounters the adduct, it may stall or become blocked. If this situation occurred, only 13 base pairs of the duplex would remain, which may melt spontaneously at 37 °C. Hence, the lack of extensive WRN helicase inhibition when translocating on the adducted strand of substrates 7–10 may not reflect helicase unwinding past the adduct. To reduce the possibility of spontaneous fraying of the partially unwound DNA substrate, WRN helicase reactions were repeated at a temperature of 30 °C, which is 6 °C below the theoretical Tm of the 13-bp duplex calculated from G/C content and salt concentration. Under these conditions, helicase activity by WRN (4.6 nM) showed little or no inhibition with substrates 7–9 and ~2-fold inhibition with substrate 10 (supplemental Fig. 1). Similar results were obtained using 2.3 nM WRN (data not shown). Extents of unwinding of the adducted substrates relative to control at 30 °C were comparable with those observed at 37 °C. This would suggest that duplex unwinding is due to the helicase action. Overall, these results indicate that for intercalated BaP-dA adducts residing in the translocating strand, cis-opened adducts are slightly more inhibitory than trans-opened adducts. However, the extent of inhibition (<2-fold) was not that dramatic for any of the substrates with translocating strand BaP-dA adducts, trans or cis. Available NMR data suggest that these two types of adducts, unlike cis- versus trans-opened dG adducts, produce qualitatively similar perturbations in the DNA structure. The observed inhibition of WRN helicase activity by the BaP DE-dA adducts was strand-specific, since neither class of BaP dA adducts residing in the strand opposite to the one on which WRN translocates hindered WRN helicase activity.


Figure 3
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  		FIGURE 2.
Effects of BaP DE-dA adducts on WRN helicase activity. A, effects of BaP DE-dA adducts situated in the strand that WRN displaces. Reaction mixtures (20 µl) containing 10 fmol of the indicated forked duplex DNA substrate and indicated concentrations of WRN were incubated at 37 °C for 15 min. Products were resolved on native 12% polyacrylamide gels. Percentage of unwinding (the mean value of at least three experiments) is shown with the S.D. indicated by error bars, as is the case for the remaining figures. B, effects of BaP DE-dA adducts situated in the strand on which WRN translocates.

 


Figure 4
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  		FIGURE 3.
Effects of BaP DE-dG adducts situated in the strand that WRN displaces. Reaction conditions are the same as in Fig. 1.

 
Effect of BaP DE-dG Adducts on WRN Helicase Activity—We were next interested in observing the effects of BaP DE-dG adducts on WRN helicase activity, since these adducts, depending on their stereochemistry, have markedly different effects on B-form double-stranded DNA structure compared with the BaP DE-dA adducts. We first tested helicase substrates with the single BaP DE-dG adduct positioned centrally in the duplex tract on the strand opposite to the one WRN translocates (substrates 11–15). For all of the five substrates tested, the percentage of substrate unwound was dependent on the WRN concentration (Fig. 3). Relative to the unadducted substrate 11, WRN unwinding was not affected by the BaP DE-dG adducts residing in the nontranslocating strand, regardless of its stereochemistry.


Figure 5
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  		FIGURE 4.
Effects of BaP DE-dG adducts situated in the strand on which WRN translocates. Reaction conditions are the same as in Fig. 2. A, phosphor images of typical gels. Shown for each gel is a no enzyme control (NE) (lane 1), 0.575 nM WRN (lane 2), 1.15 nM WRN (lane 3), 2.3 nM WRN (lane 4), 4.6 nM WRN (lane 5), and heat-denatured DNA substrate control (lane 6). B, percentage unwinding.

 
We next tested substrates with a single BaP DE-dG adduct situated on the strand on which WRN translocates (substrates 16–20). As before, the percentage of duplex unwound depended on the concentration of WRN present in the reaction (Fig. 4). Significant inhibition of WRN helicase activity by all four BaP DE-dG adducts tested was observed at WRN protein concentrations of 1.15, 2.3, and 4.6 nM WRN, where there was appreciable unwinding of the unadducted forked duplex substrate. The extent of helicase inhibition was not dependent on the stereochemistry of the specific adduct, since the percentage of substrate unwound at each WRN concentration was not significantly different for each of the adducted substrates. Clearly, all four BaP DE-dG adducts were able to potently block WRN unwinding, achieving a maximal level of ~6-fold inhibition at a WRN concentration of 2.3 nM, in which the unadducted substrate 16 was unwound 50% (Fig. 4B). WRN exonuclease activity at the blunt end of the unadducted or adducted substrates was minimal, as evidenced by the appearance of an intact released oligonucleotide on native gels (Fig. 4A) and confirmed by analysis of products on urea denaturing gels (data not shown). Taken together, the results indicate that WRN unwinding is inhibited in a strand-specific manner by BaP DE-dG adducts; however, the stereochemistry or orientation of the adduct did not have a significant effect on the extent of inhibition.

WRN Is Not Sequestered by the BaP DE-dG Modified DNA—Certain helicases have been found to be sequestered by DNA molecules harboring various types of covalent lesions (for a review, see Ref. 56). If sequestration of WRN occurs by the covalent BaP adduct, preincubation of WRN with the unlabeled single-stranded DNA or forked duplex DNA containing the adduct should trap the WRN helicase and prevent it from unwinding a radiolabeled, unadducted forked duplex tracker substrate. Since WRN is profoundly inhibited by the trans-R dG adduct situated on the strand on which WRN translocates, we chose this substrate to examine for potential sequestration of the WRN helicase.

When WRN was preincubated with either the single-stranded unadducted oligonucleotide S or trans-R-dG adducted oligonucleotide U, little to no inhibition of WRN helicase activity on the tracker substrate was observed at any level of the oligonucleotide tested (Fig. 5, A and B). However, both the 16-unadducted and 18-trans-R forked duplex DNA inhibited unwinding of the tracker substrate (Fig. 5, C and D). The ability of forked duplex but not single-stranded DNA to inhibit WRN helicase activity on a tracker substrate is consistent with previous results of sequestration assays with DNA molecules of different sequences (57), indicating that WRN preferentially interacts with the forked duplex compared with single-stranded DNA. As seen in Fig. 5E, since there was little to no difference in the extent of unwinding of the tracker substrate at each level of the adducted and unadducted substrates, this result suggests that WRN was not preferentially sequestered by the forked duplex molecule bearing the trans-R DE-dG adduct relative to unadducted DNA.


Figure 6
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  		FIGURE 5.
Sequestration of WRN on the BaP DE-modified or unmodified DNA molecules. Reaction mixtures (20 µl) containing 3 nM WRN (lanes 2–9) and increasing amounts of indicated oligonucleotide S (A), oligonucleotide U (B), forked duplex 16-unadducted (C), or forked duplex 18-trans-R (D) (lane 2, 0 fmol; lane 3, 12.5 fmol; lane 4, 25 fmol; lane 5, 50 fmol; lane 6, 100 fmol; lane 7, 200 fmol; lane 8, 250 fmol; lane 9, 500 fmol) were incubated at 37 °C for 3 min. After 3 min, 10 fmol of radiolabeled tracker forked duplex substrate was added to each reaction and incubated for an additional 7 min at 37 °C. Helicase reaction products were resolved on 12% polyacrylamide gels. No enzyme (lane 1) and heat-denatured DNA substrate (lane 10) controls are shown for each gel. E, percentage unwinding of the tracker substrate is shown as a function of the amount of competitor DNA in the reaction.

 


Figure 7
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  		FIGURE 6.
Effect of BaP DE-dG adducts on RECQ1 helicase activity. Reaction mixtures (20 µl) containing 10 fmol of the indicated forked duplex DNA substrate and the indicated concentrations of RECQ1 were incubated at 37 °C for 15 min. The percentage of unwinding is shown.

 
Effects of BaP DE-dG Adducts on RECQ1 and UvrD Helicase Activities—The inhibition of WRN helicase activity by the trans-S-BaP and trans-R-BaP dG adducts positioned in the strand on which WRN translocates raised the question of whether other 3'–5' DNA helicases would be similarly affected or whether the inhibition was unique to WRN. Therefore, we tested RECQ1 helicase, another human enzyme that is a member of the RecQ family (superfamily 2), and Escherichia coli UvrD, a member of Superfamily 1, on selected DNA substrates used for the WRN helicase characterization.

Throughout the RECQ1 concentration range (2.5–20 nM), the unwinding activity catalyzed by RECQ1 helicase was inhibited for either adducted substrate compared with the unadducted substrate (Fig. 6). In the presence of 5 nM RECQ1, 39% of the 16-unadducted substrate was unwound, compared with only 2.6 and 4.7% of the 17-trans-S and 18% trans-R substrates, respectively (Fig. 6). In the presence of 10 nM RECQ1, 77% of the 16-unadducted substrate was unwound by RECQ1, whereas only 10% 17-trans-S substrate and 20% trans-R substrate was unwound (Fig. 6). At a 2-fold higher concentration of RECQ1 (20 nM), unwinding of the 17-trans-S and 18-trans-R substrates was only slightly greater compared with the level of unwinding catalyzed by 10 nM RECQ1, indicating that increasing RECQ1 helicase concentration does not fully overcome the inhibition exerted by these adducts.

We next tested the ability of UvrD to unwind the 17-trans-S and 18-trans-R helicase substrates (Fig. 7A). As evidenced by the quantitative data, the -fold inhibition of UvrD helicase activity was dependent on the concentration of UvrD enzyme. For example, at 0.38 nM UvrD, 31% of the 16-unadducted substrate was unwound compared with 5.5 and 8.5% of the 17-trans-S and 18-trans-R substrates, respectively (Fig. 7A). At 1.5 nM, UvrD unwound 22 and 40% of the 17-trans-S and 18-trans-R substrates, respectively, compared with 83% of the 16-unadducted substrate (Fig. 7A). However, as UvrD concentration was increased, progressively more of either adducted substrate was unwound. At 3 nM UvrD, 44 and 68% of the 17-trans-S and 18-trans-R substrates, respectively, were unwound. At 6 nM UvrD, 77 and 90% of the 17-trans-S and 18-trans-R substrates, respectively, were unwound. These results indicate that although UvrD is effectively inhibited by the trans-R or trans-S dG BaP adducts at low UvrD concentrations, the inhibition can be overcome by increasing the UvrD concentration. A kinetic analysis of DNA unwinding by 12 nM UvrD demonstrated similar rates of helicase activity for the 16-unadducted, 17-trans-S, and 18-trans-R substrates (Fig. 7B), indicating that inhibition of UvrD helicase activity by the trans-S or trans-R dG BaP adduct is not observed at higher UvrD concentrations.


Figure 8
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  		FIGURE 7.
Helicase inhibition by a BaP DE-dG adduct is overcome at higher enzyme concentrations of UvrD. Reaction mixtures (20 µl) containing 10 fmol of the indicated forked duplex DNA substrate and indicated concentrations of UvrD (A) or WRN (C) were incubated at 37 °C for 15 min. The percentage of unwinding is shown. B, reaction mixtures (120 µl) containing a 0.5 nM concentration of the indicated forked duplex DNA substrate (filled circle, 16-unadducted; filled square, 17-trans-S; filled triangle, 18-trans-R) and 12 nM UvrD were incubated at 37 °C for the indicated time periods. The percentage of unwinding is shown.

 
The ability of UvrD to unwind the adducted substrates at higher enzyme concentrations may occur due to the enzyme binding to both the forked and blunt ends of the DNA substrate, since UvrD is known to unwind blunt duplexes at higher concentrations (57). To address this possibility, we tested UvrD unwinding of a 22-bp blunt duplex corresponding to the double-stranded region of substrate 16 under the conditions used for the UvrD helicase assays with the adducted substrates. The results from these experiments, shown in supplemental Fig. 2, demonstrate that UvrD is able to unwind greater percentages of the blunt duplex substrate with increasing UvrD concentrations. These results are compared with UvrD unwinding of 16-unadducted, 17-trans-S, and 18-trans-R substrates. This comparison suggests that the ability of UvrD to overcome the inhibition by these adducts is at least partially due to unwinding from the blunt end of these substrates.

The ability of UvrD to overcome the helicase inhibition by the BaP DE-dG adduct at higher UvrD concentrations raised the possibility that WRN might behave similarly. To address this, we tested a 2-fold higher concentration of WRN helicase, 9.2 nM, on unwinding of the 18-trans-R substrate. As shown in Fig. 7C, only a slight increase in unwinding of the adducted substrate, ~1.7-fold, was observed at 9.2 nM WRN compared with 4.6 nM WRN. However, the level of unwinding of the adducted substrate by WRN was still ~3.5-fold less than that of the unadducted substrate.

Effect of RPA on WRN-catalyzed Unwinding of BaP DE-dG-adducted Substrates—The human single-stranded DNA binding protein RPA physically and functionally interacts with WRN helicase, enabling the enzyme to unwind long DNA duplexes efficiently. The functional interaction between WRN helicase and RPA may also be important to enable the enzyme to unwind damaged DNA substrates. To address this possibility, we investigated the effect of RPA on WRN unwinding of the 18-trans-R- and 20-cis-R-adducted substrates by performing kinetic assays on these and the unadducted substrate in the presence or absence of RPA. The adducted substrates chosen were those that were most poorly unwound by WRN in the absence of RPA in the 15-min reaction (Fig. 4).

The kinetics of unwinding of the unadducted substrate by WRN were linear throughout the 4-min time course, yielding an initial rate of 1.5 bp/min DNA substrate unwound (Fig. 8A). The 18-trans-R and 20-cis-R substrates were unwound very inefficiently by WRN, resulting in only 0.17 bp/min and 0.37 bp/min DNA substrate unwound, respectively (Fig. 8, B and C). This corresponded to a 9-fold and 4-fold reduction in rates for modified substrates 18-trans-R and 20-cis-R, respectively, compared with the unadducted substrate. In the presence of RPA (12 nM), WRN helicase activity was significantly increased on the unadducted substrate, yielding an initial rate of unwinding of 7.3 bp/min over the first 1 min of the time course (Fig. 8A). For the 18-trans-R substrate, the presence of RPA also significantly increased WRN helicase activity to a rate of 6.6 bp/min substrate unwound during the first min of the time course (Fig. 8B). A comparison of the results revealed that the rate of WRN helicase activity in the presence of RPA on the adducted substrate was significantly increased by ~39-fold compared with the rate of unwinding by WRN acting alone on this substrate in the absence of RPA; moreover, WRN helicase activity on the 18-trans-R substrate was only 10% less than that of the WRN unwinding rate for the unadducted substrate when RPA was present in the reaction. These results indicate that RPA enables WRN to overcome the inhibition of unwinding by the trans-R BaP DE-dG adduct. Similar, but not quite as dramatic, results were obtained from experiments with the 20-cis-R substrate (Fig. 8C). WRN helicase activity on 20-cis-R was increased from 0.37 to 5.5 bp/min when RPA was present, a 19-fold elevation in the linear rate. An important conclusion from these studies is that RPA greatly enhances WRN helicase activity on substrates with a centrally located DNA lesion that the helicase acting alone unwinds poorly. The stimulatory effect of RPA on WRN helicase activity is evident for substrates harboring a DNA lesion that occupies the minor groove or causes the adducted base and its opposing base to flip out, suggesting that the WRN-RPA functional interaction can persist in the face of various DNA structural perturbations to the double helix.


Figure 9
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  		FIGURE 8.
RPA enables WRN helicase to unwind efficiently the BaP DE-dG adducted DNA substrates. Reaction mixtures (120 µl) containing a 0.5 nM concentration of the indicated forked duplex DNA substrate (A, 16-unadducted; B, 18-trans-S; C, 20-cis-R), 4.6 nM WRN, and the presence (filled circle) or absence (open circle) of 12 nM RPA were incubated at 37 °C for the indicated time periods. The percentage of unwinding is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The unique structural motifs presented by a series of DNA adducts of BaP DEs provided an opportunity to study the importance of WRN helicase contacts with the DNA double helix during the course of unwinding when it encounters specific types of DNA damage. Since the stereochemistry and base to which the BaP DE adduct is covalently bound dictate the nature of the helix perturbation, we were able to assess the effects of defined DNA structural motifs on helicase-catalyzed DNA unwinding. The substrates with a single BaP DE-dA adduct situated on the strand opposite to that on which WRN translocates did not inhibit WRN helicase activity. Surprisingly, we observed only modest inhibition by the cis or trans BaP DE-dA adduct positioned in the helicase-translocating strand. Previously, we reported that duplex DNA unwinding catalyzed by the WRN helicase is inhibited by benzo[c]-phenanthrene (BcPh) DE-dA adducts in a strand-, orientation-, and stereoisomer-dependent manner (58). The aromatic portions of the 1R and 1S trans-opened BcPh DE N6-dA adducts used in that study intercalate between base pairs in duplex DNA with some local stretching of the double helix to accommodate the intercalated aromatic ring. The nonaromatic ring of the trans BcPh adduct is situated in the major groove, whereas the intercalated aromatic portion projects toward but not into the minor groove. The profound inhibition of unwinding by BcPh adducts provided evidence that a bulky, intercalating covalent adduct can have major consequences for WRN helicase function. However, the results from the current study suggest that for intercalating BaP DE-dA adducts, local double helix perturbations of buckling, twisting, or underwinding do not significantly deter the WRN helicase reaction. Thus the effect of a given bulky covalent DNA adduct on helicase function may depend on its chemical nature and how the adduct resides in the DNA double helix. Notably, BcPh DE dA adducts have no effect on the thermal stability (Tm) of DNA duplexes (59), whereas BaP DE-dA adducts result in substantially lowered Tm values relative to the corresponding unadducted duplexes (60). This observation indicates that BcPh DE dA adducts do not destabilize the DNA duplex, whereas the corresponding BaP adducts do so. Partial unwinding at the site of BaP DE dA adducts could facilitate the action of helicase, thereby possibly overcoming any unfavorable steric effects of the bulk of the adducts.

In contrast to the results with the BaP DE-dA adducts, either cis or trans BaP DE-dG adducts positioned in the helicase-translocating strand exerted profound inhibition of WRN helicase activity, whereas the same adducts positioned in the opposite strand to the one on which WRN translocates did not inhibit WRN unwinding of the substrate. These results indicate that WRN helicase activity is strongly inhibited in a strand-specific manner by two conformationally very different types of adducts: cis BaP DE-dG adducts, which cause a flipped out base pair, and trans adducts, which reside in the minor groove. Similar to WRN, human RECQ1 helicase was profoundly inhibited by a BaP DE-dG adduct in a strand-specific manner, suggesting that RecQ helicases may be similarly affected by other types of DNA damage. A possible mechanism for the profound inhibition of WRN or RECQ1 helicase activity by the minor groove BaP DE-dG adduct is suggested by a recently identified conserved helix-turn-helix motif found in RecQ helicases that was shown to mediate minor groove binding in the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (61).

Consistent with the idea that WRN helicase activity is particularly susceptible to minor groove perturbation, we demonstrated previously that WRN-catalyzed DNA unwinding is potently inhibited by the minor groove-binding drugs distamycin A and netropsin as compared with other DNA-binding drugs (62). The adverse effects of biologically relevant DNA modifications, either covalent or noncovalent, on the catalytic activities of WRN and other RecQ helicases may be relevant to the genetic damage and cell transformation induced by the adducts and/or the mechanism of action of chemotherapeutic drugs such as distamycin analogs that position themselves in the minor groove.

From the helicase protein titration studies, we observed that the inhibition of DNA unwinding by the minor groove adduct on the helicase-translocating strand was observed at all concentrations of WRN or RECQ1 helicase tested. Despite the effective inhibition of WRN helicase activity by a single BaP DE-dG adduct in the strand along which the helicase translocates, the adducted forked duplex molecule did not trap WRN to any greater extent than the unadducted molecule. Coupled together with the observation that WRN does not preferentially degrade by exonuclease digestion the poorly unwound adducted substrate, the results suggest that WRN dissociates as rapidly from the BaP-modified DNA molecule as the unadducted DNA molecule.

RPA, a single-stranded DNA binding protein that is implicated in the processes of DNA replication, repair, and recombination, was shown to specifically stimulate WRN helicase unwinding of long DNA duplexes. For the first time, we demonstrate that the presence of RPA can enable WRN to overcome the inhibition of DNA unwinding exerted by a single covalent base adduct residing in the translocating strand within the duplex. Understanding the biological significance of the WRN-RPA interaction in the replicational stress response may be important to deciphering the DNA metabolic defects in WS. WS cells have a prolonged S phase (11), asymmetry of DNA replication fork progression (63), slower rate of repair associated with DNA damage induced in S-phase, reduced induction of RAD51 foci, and a higher level of strand breaks (64). The in vivo evidence implicating WRN in the recovery of DNA synthesis after replication arrest poses the question of whether WRN functions together with RPA in a critical step to resolve a key replication or recombinational intermediate that arises from fork stalling or collapse. RPA has been found to co-localize with WRN upon replication arrest (65) and DNA damage (66), suggesting that the two proteins may indeed collaborate to perform certain cellular function(s). As a component of the replication stress response, RPA may serve to enable WRN helicase to overcome DNA-blocking base lesions introduced by exposure to exogenous DNA-damaging agents such as benzopyrene or DNA damage introduced by endogenous biochemical processes, such as oxidation. A role for WRN in conferring resistance to the lesions N3-methyladenine and O6-methylguanine was recently suggested by cellular studies (67); however, the importance of WRN helicase function remains to be shown. In addition to base modifications, RPA may also enable WRN to unwind DNA substrates with alterations to the sugar phosphate backbone, as recently suggested by in vitro WRN helicase studies with DNA substrates that contain synthetic vinylphosphonate modifications (68). By its partnership with RPA, WRN may be able to overcome helicase inhibition and ensure normal DNA transactions despite the presence of a potentially mutagenic lesion.

In the absence of functional WRN protein, the rate of replication fork elongation emanating from individual bidirectional origins is abnormal, leading to asymmetric fork progression (63). One reason for the observed slower rates of replication fork movement and asymmetry of replication fork progression in WS cells compared with normal cells may be the inability to replicate past lesions in the template or to restart a stalled replication fork (63). This rationale supports a role of WRN in unwinding double-stranded DNA to promote fork progression when it encounters a blocking lesion. If this model is true, our results would suggest that WRN functions with RPA to unwind past certain covalent adducts in the template strand. To generate RPA-coated single-stranded DNA necessary to activate the ATR-dependent DNA damage response pathway in response to genotoxic lesions, a mechanism of decoupling helicase and polymerase activities at a replication fork was very recently elucidated (69). The evidence that WS cells have a defect in replication fork progression that is more pronounced at certain genomic sites compared with others (63) may be a consequence of the absence of an appropriate signal mediated by the WRN-RPA interaction at a stalled replication fork.


   FOOTNOTES
 
* This research was supported by the Intramural Research Program of the NIA and NIDDK (National Institutes of Health). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http:www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 410-558-8578; Fax: 410-558-8157; E-mail:broshr{at}grc.nia.nih.gov.

3 The abbreviations used are: WRN, Werner; WS, Werner syndrome; RPA, replication protein A; BaP, benzo[a]pyrene; DE, diol epoxide; BcPh, benzo[c]phenanthrene. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Steven Matson (University of North Carolina at Chapel Hill) and Dr. Mark Kenny (Albert Einstein Cancer Center) for kindly providing purified recombinant UvrD helicase and RPA heterotrimer, respectively. We thank Drs. Sudha Sharma and Wen-Hsing Cheng (Laboratory of Molecular Gerontology, NIA, National Institutes of Health) for critical reading of the manuscript.



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R. M. Brosh Jr and V. A. Bohr
Human premature aging, DNA repair and RecQ helicases
Nucleic Acids Res., December 3, 2007; 35(22): 7527 - 7544.
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