Unwinding of a DNA Triple Helix by the Werner and Bloom Syndrome Helicases*

Robert M. Brosh Jr.Dagger , Alokes MajumdarDagger , Shital DesaiDagger , Ian D. Hickson§, Vilhelm A. BohrDagger , and Michael M. SeidmanDagger

From the Dagger  Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore, Maryland 21224 and § Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom

Received for publication, July 28, 2000, and in revised form, November 2, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3' right-arrow 5' polarity, and require a 3' single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3' tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5' tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Despite the obvious importance of genetic stability, the mammalian genome has an abundance of sequences that are potentially destabilizing. Elements that can form non-duplex structures, such as DNA triple helices (1, 2), G quartets (3-5), hairpins (6, 7), and cruciforms (8), all have the capacity to interfere with transcription and replication. Moreover, many studies have described the role of these elements in DNA rearrangements such as deletions, sister chromatid exchanges, homologous and illegitimate recombination events, etc. (reviewed in Refs. 9 and 10). With such an array of provocative sequence elements, it seems likely that cells would have developed the capacity for controlling the potential of these sequences for genome destabilization.

Some insight into the nature of the enzymology involved in the maintenance of genetic integrity has come from the study of two "genome instability" disorders, Bloom and Werner syndrome. Patients with Werner syndrome (WS)1 are characterized by premature aging (11) and a high incidence of certain cancers. They have elevated frequencies of spontaneous deletion mutations in the HPRT gene (12) and show a variety of karyotypic abnormalities including inversions, translocations, and chromosomal losses (13, 14). The mutant gene in WS has been identified as a member of the RecQ helicase family, and the protein is a 3'-5'-helicase (15, 16) and a 3'-5'-exonuclease (17-19). A role in replication is implied by the demonstration that the WRN helicase interacts with and is stimulated by the human replication protein A (RPA) (20, 21). The protein has been recovered from cells in a replication complex (22), and the Xenopus homologue, FFA-1, is required for the formation of replication foci (23). In addition, recent work (24-26) suggests a role for the WRN helicase in telomere maintenance. Finally, the chromosomal instability of WS cells suggests that the enzyme plays an anti-recombinogenic role in wild type cells.

Bloom syndrome (BS) patients display a variety of symptoms, among them growth retardation, immunodeficiency, sun sensitivity, and a marked propensity to develop cancers of all varieties (27-29). At the cellular level there is an increased frequency of spontaneous mutation (30) and somatic recombination (31). Elevated levels of sister chromatid exchanges have long been associated with the disorder (32). As with Werner syndrome there is an enhanced frequency of large deletion mutations in the HPRT gene (33). The mutant gene in Bloom syndrome patients also encodes a RecQ family helicase with 3'-5' polarity although it lacks an exonuclease activity (34-36). It is found associated with other proteins involved in DNA metabolism (37), including RPA (38). The marked chromosomal instability of BS cells and recent biochemical evidence (39) argue for an anti-recombinogenic activity for the wild type BLM protein as well.

Whereas the defining function of these and all other helicases is the unwinding of duplex nucleic acid, recent work indicates that the BLM and WRN enzymes are active on alternate DNA structures. In particular, they can unwind tetrahelical structures that can form in stretches of G-rich DNA (G DNA) (40, 41). Such sequences are widely distributed in the genome (5) and are found, among other places, at telomeres (42-44) and in the triplet repeats such as the CGG associated with Fragile X syndrome (45). They can form under physiological conditions and inhibit DNA synthesis in vitro (46). It has been suggested by many authors that these complexes, appearing as the result of single strand exposure during replication, recombination, or transcription, could play a causal role in genomic rearrangements, including strand exchange, strand slippage, and the size changes seen in the triplet expansion disorders. The possibility that the genomic instability of the WS and BS disorders might be due to a failure to resolve alternate DNA structures has generated considerable interest (47).

The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome (1). Triplexes have been known for many years (Ref. 48 and reviewed in Refs. 49 and 50) and are generated when a third strand lies in the major groove of duplex DNA. They occur most readily on polypurine:polypyrimidine sequences, and the third strand may be composed of either pyrimidines or purines, with the most stable resultant structure dictated by the specific sequence (51). They can form when an appropriate sequence partially melts with one of the single strands folding back to complex with the adjacent duplex (52) and have been demonstrated in chromosomes and nuclei (53, 54). These complexes can also be formed by oligonucleotides directed against specific target sequences. This has prompted their consideration as gene-targeting reagents (55-58).

Although there is a 4-decade literature on triple helices and a considerable interest in the role of helicases in DNA transactions, there have been remarkably few studies on the activity of these enzymes on triplexes. The DDA protein of phage T4 can unwind a triplex (59), as can the SV40 T antigen (60). However, there has been no report of the activity of human helicases with these substrates. In this report we describe the activity of the BLM and WRN helicases on a pyrimidine-purine:pyrimidine triple helix and corresponding duplex structures.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins-- Recombinant hexahistidine-tagged human WRN protein was overexpressed in Sf9 insect cells and purified as described elsewhere (20). Recombinant hexahistidine-tagged human BLM protein was overexpressed in Saccharomyces cerevisiae and purified as previously described (61). UvrD (DNA helicase II) was kindly provided by Dr. Steven Matson (University of North Carolina, Chapel Hill). T4 polynucleotide kinase was obtained from New England Biolabs.

Oligonucleotides, Nucleotides, and DNA-- The following oligonucleotides were prepared: TC30, 5'-TCTTTTTCTTTCTTTTCTTCTTTTTTCTTT; TC30 5' tail, 5'-TGACGCTCCGTACGA-TC30; TC30 3' tail, 5'TC30-TCACGCTCCGTACGA. The cytosines in the TC30 portion were 5 methylcytosines. This modification stabilizes the triplexes at physiological pH (62). The 28-mer oligonucleotide 5'-TCCCAGTCACGACGTTGTAAAACGACGG-3' was from Life Technologies, Inc. M13mp18 ssDNA was from New England Biolabs. Ribonucleotides, deoxyribonucleotides, and ATPgamma S were from Roche Molecular Biochemicals.

Triplex Substrates-- The plasmid psupF5 contains a duplex sequence that serves as a target for TC30 and related oligonucleotides. Cleavage of the plasmid with NdeI released fragments of 4 and 0.6 kilobase pairs. The triplex site lies 1800 bases from one end of the large fragment. Triplexes were prepared by incubation of 3 pmol of 5'-32P-labeled oligonucleotide (based on the molecular weights of the reaction components) overnight at room temperature with 6 pmol of NdeI-cleaved plasmid in a buffer containing 33 mM Tris acetate, pH 5.5, 66 mM KOAc, 100 mM NaCl, 10 mM MgCl2, and 0.1 mM spermine. The complexes were then separated from unbound oligonucleotide by gel filtration chromatography using Bio-Gel A-5M resin. The 6-base pair duplex extension substrate was prepared as above by incubation of the 3'-tailed third strand with plasmid that was cleaved with BglII and filled in by incubation with Klenow polymerase. This construction placed the triplex at the extreme end of the duplex DNA with a 6-base pair duplex extension. The 3'-tailed "blunt triplex" was prepared by incubation of a 30-mer duplex oligonucleotide with the 3'-tailed third strand. This duplex contained the triplex region without extensions.

Tm Determination-- A triplex was prepared by incubation of the 3'-tailed third strand with a duplex 30-base pair oligonucleotide consisting of the triplex target sequence. The triplex was diluted into the buffer used for the helicase assays (see below), and the thermal stability was determined in a PerkinElmer Life Sciences spectrophotometer with a Peltier controlled thermal unit attached.

Restriction Protection-- The triplex target site overlaps an XbaI site, which is blocked by triplex formation. 2 µg of psupF5 were incubated overnight with 2 µM of the 5', or 3', or blunt TC30 oligonucleotides in the buffer described above. The complexes were then separated from free oligonucleotide by ethanol precipitation from 2.5 M ammonium acetate. They were suspended in 50 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM spermine, 100 µg/ml bovine serum albumin and digested with XbaI and PstI at 37 °C. Although in these experiments the triplexes were formed on circular plasmids prior to restriction digestion, triplex formation was equally efficient on linear DNA.

Duplex Helicase Substrates-- To prepare the TC30 duplex DNA substrate, the TC30 3' tail oligonucleotide was 5'-end-labeled and annealed to a 30-mer oligonucleotide complementary to TC30 (TC30'). The annealing mixture contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 pmol TC30', and 4 pmol of 32P-labeled TC30. The annealing mixture was incubated at 90 °C for 5 min and allowed to cool to room temperature over the course of 3 h. The forked duplex substrate was prepared by annealing a 50-mer that contained the 30-base complement of TC30 and a 20-base noncomplementary 5' tail. The labeled annealed DNA duplexes were electrophoresed on a 12% polyacrylamide (19:1, acrylamide:bisacrylamide) 1× Tris borate, EDTA gel, and the duplex fragment was excised. The DNA fragment was purified using the QIAEX II polyacrylamide gel extraction kit (Qiagen) and eluted with 10 mM Tris-HCl, pH 8.0. The 28-base pair M13mp18 partial duplex substrate was constructed with a 28-mer complementary to positions 6296-6323 in M13mp18. The 28-base pair M13mp18 partial duplex substrates were constructed as described previously (63) with the following modifications. The 28-mer oligonucleotides were labeled at their 5' ends using T4 polynucleotide kinase and [gamma -32P]ATP and annealed to M13mp18 single-stranded DNA circles. Partial duplex DNA substrates were purified by gel filtration column chromatography using Bio-Gel A-5M resin (Bio-Rad).

Helicase Assays-- Helicase assay reaction mixtures (20 µl) contained 40 mM Tris borate, pH 7.4, 5 mM MgCl2, 5 mM dithiothreitol, 2 mM ATP (or the indicated nucleoside 5' triphosphate), and the indicated amounts of WRN or BLM helicase. The amount of the TC30 triplex or 3'-tailed duplex molecules in the reaction mixtures was ~15 fmol. The amount of the M13 partial duplex molecules was 4 fmol. Reactions were initiated by the addition of WRN or BLM protein and incubated at 24 °C for 30 min. At the end of the incubation, a 10-µl aliquot of loading buffer (40% glycerol, 0.9% SDS, 0.1% bromphenol blue, 0.1% xylene cyanol) was added to the mixture, and the products of helicase reactions were resolved on nondenaturing polyacrylamide gels (12% acrylamide, 40 mM Tris acetate, pH 5.5, 5 mM MgCl2, 25% glycerol) at 4 °C. The 30-mer 3'-tailed "blunt triplex" substrate was resolved on a 10% polyacrylamide gel containing 5% glycerol. An excess of unlabeled third strand was added to the reaction mixtures at the end of the reaction to preclude reassociation of the unwound labeled third strand with the duplex. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager or film autoradiography and quantitated using the ImageQuant software (Molecular Dynamics). The percent helicase substrate unwound was calculated by the following formula: % Displacement = 100 × P/(S P). P is the product and S is the substrate. The values for P and S have been corrected after subtracting background values in the no enzyme and heat-denatured controls, respectively. Helicase data shown are representative of at least three independent experiments.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Enzyme Preparations-- The recombinant WRN and BLM proteins were characterized by standard helicase and ATPase assays (not shown). The activity of the WRN enzyme with a duplex substrate, formed by the hybridization of a 28-base oligonucleotide to single strand circular M13, was measured. This substrate was almost completely unwound at higher concentrations of enzyme (30-60 nM) (data not shown). Unwinding of the M13 partial duplex by the BLM enzyme was virtually complete at enzyme concentrations >= 16 nM. The specific ATPase activities of WRN and BLM enzymes using M13mp18 single-stranded DNA as the DNA effector were determined to be 219 ± 14 and 1163 ± 358 min-1, respectively, consistent with previously published values (35, 64).

Triplex Substrates-- We prepared triple helices with a pyrimidine motif third strand with 15 nucleotide 3' or 5' single strand tails or with no tail. The sequences of the target duplex and the third strands are shown in Fig. 1A. These triplexes were stable at physiological pH. In the buffer used for the helicase assays the Tm of the TC30 triple helix was 57.9 °C, and the Tm of the underlying duplex was 62.9 °C. Duplex unwinding by both helicases is dependent on a single strand 3' tail. All the triplexes used in these experiments were based on blunt duplexes, eliminating the possibility that triplex unwinding was due to the disruption of the underlying duplex.



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Fig. 1.   Triplex DNA substrates used in these experiments. A, the sequence of the TC30 oligonucleotide and the different triplex substrates. The target was embedded in the supF5 plasmid (flanking sequences indicated as - - -). B, restriction enzyme protection by triplex formation. Triplexes were formed with the 3' tail (lane 1), the blunt (lane 2), or 5' tail (lane 3) oligonucleotide on the supF5 plasmid with the triplex target. These complexes, as well as the supF5 plasmid without an oligonucleotide (lane 4) or the supF12 plasmid, which lacks the TC30 triplex site, with the 3'-tailed oligonucleotide (lane 5), were digested with PstI and XbaI. Plasmid digested with PstI alone is also shown (lane 6). Triplex formation protects the XbaI site. An arrow denotes the 4000-base pair fragment with the triplex site. 0.5 µg of each sample was loaded on each lane. A HindIII digest of phage lambda DNA was run as a molecular weight marker.

Restriction Site Protection by Triplex Formation-- Triplex formation on the target sequence in psupF5 partially occludes an XbaI site. As shown in Fig. 1B protection of the site from XbaI cleavage could be demonstrated following triplex formation with the oligonucleotides, whereas cleavage by PstI, whose sites are not near the triplex target, was unaffected. Another plasmid (psupF12), which does not have the TC30 triplex target sequence, was also incubated with the TC30 oligonucleotides. However, as expected, there was no protection from XbaI digestion.

Activity of WRN and BLM Helicase on Triplexes with Different Third Strands-- WRN and BLM helicases are most active on duplex DNA with 3' single strand tails (16, 40).2 We asked if the helicases would be active with DNA triplexes with a 3', 5', or no single strand tail as depicted in Fig. 1A. Each substrate was incubated with increasing amounts of the individual enzymes. As shown in Fig. 2A, the WRN was active against the 3' tail triplex with increasing amounts of triplex unwinding as a function of enzyme concentration. The extent of unwinding reached a plateau at 28 nM protein with 50% of the 3' tail substrate unwound. There was no significant unwinding of the blunt and 5' tail substrates.



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Fig. 2.   Unwinding of triplex DNA by WRN (A) and BLM (B) helicases. A triplex DNA substrate with a 3' single-stranded tail (filled circles), 5' single-stranded tail (open circles), or no tail (×) was incubated with the indicated concentrations of WRN protein under the standard helicase conditions described under "Materials and Methods." Incubation was at 24 °C for 30 min. The inset shows the gel pattern with the 3'-tailed substrate. Heat-denatured control, filled triangle.

Similar data were obtained with the BLM helicase with only the 3'-tailed substrate unwound (Fig. 2B). Unwinding was measurable at the lowest levels of enzyme, and more than 90% of the substrate was unwound at higher enzyme concentrations.

Nucleotide Preference of WRN and BLM Helicases in the Triplex Unwinding Assay-- The activity of helicases is dependent on a nucleoside triphosphate (65). We examined the nucleotide preference of the WRN enzyme in unwinding the 3' tail triplex substrate (data not shown). 21 nM enzyme was incubated with the substrate in the presence of the ribo- and deoxyribonucleoside triphosphates. Unwinding was observed with dCTP (27%), CTP (50%), dATP (50%), and ATP (26%). There was no significant activity in the presence of the other triphosphates. The BLM helicase was less discriminating as extensive unwinding (80-90%) was observed with all triphosphates with the exception of UTP (71%) and TTP (26%). When ATPgamma S was substituted for the other triphosphates there was no unwinding with either enzyme.

Triplex Unwinding by WRN and BLM Helicases Does Not Require a Fork-- The substrate employed in the preceding experiments presented different structural elements to the enzymes. These included the single strand tail, the triplex region, and the duplex adjacent to the triplex. The nature of the junction between single strand tails and duplex substrate of different helicases is well known to affect enzyme activity (66-68). We wanted to know if the activity of the BLM and WRN helicases would also be affected by the nature of the junction between the single strand and the triplex. Accordingly, we prepared two additional substrates. The first was based on the substrate used in the previous experiments. The plasmid was linearized at the BglII site (Fig. 1) and the 4-base 5' overhang filled in by Klenow polymerase. This was incubated with the 3'-tailed third strand to produce a triplex with a 6-base duplex extension. This substrate maintained a long duplex region as before, and a junction of a 3'-tailed third strand, a triplex, and a short duplex extension. Both enzymes unwound more than 85% of this substrate (Fig. 3).



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Fig. 3.   Activity of the WRN and BLM helicases on a triplex with a 6-base pair extension. Heat-denatured substrate, filled triangle.

The second substrate consisted of the 3'-tailed third strand complexed to an oligonucleotide duplex such that there were no duplex extensions beyond the triplex (3'-tailed blunt triplex). This structure was unwound by the WRN enzyme, although less efficiently than the previous substrate (50% unwound at 56 nM WRN helicase) (Fig. 4A). The BLM enzyme was more active with this substrate with 80% unwound at 32 nM enzyme (Fig. 4B). There was no activity with either enzyme in the presence of ATPgamma S. These experiments showed that both enzymes could unwind the triplex without a requirement for a single strand:double strand fork at the junction of the third strand and the triplex and without regard for the presence or absence of an associated long duplex region.



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Fig. 4.   Activity of the WRN and BLM enzymes with a triplex with no duplex extension. A, WRN helicase; B, BLM helicase.

Activity of Bloom and Werner Helicases on a Duplex With and Without a Fork-- It was of interest to know if the two enzymes would unwind a duplex substrate containing the same strand used in the triplex experiments. The 45-nucleotide fragment used as the third strand in the triplex experiments was hybridized with a 30-base oligonucleotide complementary to the triplex region. This produced a duplex with the same 3' tail as in the triplex experiments. An amount of substrate equivalent to that used in the triplex experiments ("Materials and Methods") was incubated with increasing amounts of each enzyme as before. The results of the titration with the WRN enzyme (Fig. 5A) showed little or no unwinding at concentrations that had displaced substantial amounts of triplex in the earlier experiments (compare with Fig. 2). There was detectable, but weak, unwinding by the BLM enzyme (9%) at the highest concentration of enzyme tested (64 nM) (Fig. 5B). The poor activity of the two enzymes was not a peculiarity of this particular substrate since UvrD unwound the substrate completely (not shown).



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Fig. 5.   Activity of the WRN and BLM helicases on the TC30 duplex. A TC30 duplex DNA substrate with a 3' single-stranded tail was incubated with the indicated concentrations of WRN protein (A) or BLM protein (B) under the standard helicase conditions described under "Materials and Methods." Incubation was at 24 °C for 30 min. Heat-denatured control, filled triangle.

We then asked if the same duplex could be unwound when associated with a fork rather than the single strand tail (see "Materials and Methods"). In contrast to the preceding experiment, both enzymes unwound the forked substrate, with the plateau value (85%) reached by the WRN enzyme at 7 nM and 90% unwound by the BLM enzyme at 32 nM (Fig. 6, A and B).



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Fig. 6.   Activity of WRN and BLM on a forked duplex substrate. A TC30 duplex DNA substrate with non-complementary 3' and 5' tails was incubated with the indicated concentrations of WRN (A) or BLM (B) enzyme. Heat-denatured control, filled triangle.



    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The BLM and WRN enzymes are DNA-stimulated ATPases with 3'-5'-helicase activity. They are active against short duplexes on the M13 single strand substrate, unwind longer duplex substrates in the presence of RPA (20), (38), and are potently inhibited by minor groove binding compounds such as netropsin and distamycin (69).

The structure and reaction requirements for the triplex unwinding by the WRN and BLM helicases are in accord with the previous data from experiments with duplex substrates with regard to the requirement for a 3' single strand tail (21). The nucleotide preference analysis indicated that the triplex unwinding was supported by both adenosine and cytosine triphosphates similar to that found with duplex substrates (21). The utilization of nucleoside triphosphates other than ATP or dATP has been reported for other helicases (65).

Interactions between Helicases and Substrates-- There are several models for DNA unwinding by helicases (66, 67, 70-72), based on activity with duplex substrates. In some versions helicase function is seen as an active process in which the enzyme interacts with both single- and double-stranded DNA at a junction, with the precise nature of the interactions and the subsequent events subject to much debate (67, 71). The preference of the BLM and WRN helicases for the forked duplex or the triplex substrates suggests an active engagement by the enzymes with the junction of the single strand tail and the fork or triplex. Like the BLM and WRN enzymes, a preference, if not a requirement, for a forked substrate is a feature shared by other helicases. This is often taken as an indication of a replicative role for the enzyme (66, 73-82). A possible linkage between helicase structure and substrate preference has been proposed in a recent study that suggests that the distinction between forked and non-forked substrates reflects the exclusion of the forked strand from the central channel of hexameric helicases (83). This proposal is relevant to the enzymes studied here as the BLM helicase has been shown to be a hexamer (61).

The results of our experiments with the triplex and duplex substrates can be summarized as follows: both enzymes unwound triplexes without a requirement for single strand:double strand forks at the junction of the single strand tail and the triplex region. On the other hand, the enzymes were weakly active with a nonforked duplex with a single strand tail but were both very active with the forked version of the same duplex substrate. Thus it would seem that the stimulatory influence of the fork junction seen with duplex substrates is unnecessary with the triplex substrates. This suggests that some feature of the triplex can compensate for the absence of the fork. In a triplex the third strand lies in the major groove of an intact duplex. Perhaps an interaction with the underlying duplex as well as the third strand replaces the interaction with the "other" strand at a fork. Alternatively, if the WRN enzyme, like the BLM, proves to be hexameric, then the channel exclusion hypothesis of Kaplan (83) may also explain the similar activity with the fork and triplex substrates.

Although the enhanced activity of the WRN and BLM enzymes seen with forked duplex substrates is consistent with reports on other helicases, we and others (16, 20, 21, 35, 38, 84) have observed activity by both enzymes on duplex substrates that do not have forks. With one exception (21) all these studies employed the popular M13 partial duplex substrate. The resolution of this apparent contradiction may reflect the actual DNA structure required for initiation of unwinding, as well as a particular feature of the M13 substrate. Entry into the duplex region of a tailed (not forked) substrate by BLM and WRN proteins may be dependent on the fraying of the duplex end, giving the enzyme an opportunity for a requisite interaction with the complementary strand. The M13 substrate has a long single strand "loading" region to which enzyme can be bound even if not engaged in actual unwinding. The WRN and BLM enzymes are distributive enzymes (20, 38). Should an active enzyme molecule be released from the substrate prior to initiating, then, with the M13 substrate, the released enzyme could be replaced by a one-dimensional movement of already bound enzyme. Thus the local concentration of replacement enzyme would be much greater with the M13 substrate and the probability of initiation much higher than for the short tailed duplex substrate, which would require replenishment of bound enzyme from solution. One prediction of this hypothesis is that duplex substrates with short single strand tails, the kind examined in Fig. 5, should be unwound if the enzyme concentration in solution is high enough. In experiments to be presented elsewhere, we have found that these substrates can indeed be unwound at enzyme concentrations a hundred-fold higher than required to unwind the corresponding forked substrate.3 This result demonstrates that there is not an absolute block to unwinding the tailed substrates and reconciles the apparent contradiction between the results with M13 and the tailed duplex substrates presented here.

Genomic Instability in BS and WS Cells-- Specific functions for WRN and BLM in vivo have yet to be identified, although roles in replication and as suppressors of recombination are consistent with the characteristics of cells derived from affected individuals and recent biochemical analyses (22, 23, 85). It has been proposed that these helicases unwind alternate DNA structures that might form during replication and block fork progression (37, 39, 47). In that light it is of particular interest that sequences with triplex forming potential are widely distributed in the human genome (1, 86-88). The possibility that triplexes form in vivo is supported by the reports of mammalian proteins that bind specifically to them (89-92) and to the polypyrimidine (93) and polypurine single strands (94) that would appear when the complementary strands were engaged in alternate structures.

The loss of the helicase activity of the BLM and WRN enzymes is generally seen as the basis of the genome instability that is common to both syndromes. Triplex structures are known to be recombinogenic (95, 96), and recently it has been shown that triplexes can arrest progression of Holliday junctions in a model system (97). These, like arrested replication forks, are likely to be susceptible to breakage. In light of the data presented here, it seems reasonable to suggest that triplex structures might be more persistent in WS and BS cells and could contribute to the genomic instability characteristic of both syndromes.


    ACKNOWLEDGEMENTS

We thank Dr. Nitin Puri for oligonucleotide synthesis and analysis of the thermal stability of the triplex and duplex substrates. We also thank Dr. Qin Yang for useful comments.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: LMG, Box 1, GRC, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8565; Fax: 410-558-8157; E-mail: seidmanm@grc.nia.nih.gov.

Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M006784200

2 R. M. Brosh, Jr., A. Majumdar, S. Desai, I. D. Hickson, V. A. Bohr, and M. M. Seidman, unpublished results.

3 R. M. Brosh, Jr., J. A. Sommers, C. von Kobbe, P. Karmakar, P. L. Opresko, I. I. Dianova, G. L. Dianov, and V. A. Bohr, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: WS, Werner syndrome; BS, Bloom syndrome; RPA, replication protein A; ATPgamma S, adenosine 5'-O-(thiotriphosphate).


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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