UNWINDING OF A DNA TRIPLE HELIX BY THE WERNER AND … · ABSTRACT Bloom Syndrome and Werner...

UNWINDING OF A DNA TRIPLE HELIX BY THE WERNER AND BLOOM

SYNDROME HELICASES

Robert M. Brosh Jr.1, Alokes Majumdar1, Shital Desai1, Ian D. Hickson2, Vilhelm

A. Bohr1, and Michael M. Seidman1,3

1. Laboratory of Molecular Genetics, National Institute on Aging, National

Institutes of Health, Baltimore, MD 21224

2. Imperial Cancer Research Fund Laboratories, Institute of Molecular

Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS,

United Kingdom

3. Corresponding author, LMG, Box 2, GRC, 5600 Nathan Shock Dr.,

Baltimore, MD 21224 email: [email protected], 410-558-8565,

FAX 410-558-8157

JBC Papers in Press. Published on November 10, 2000 as Manuscript M006784200 by guest on A

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ABSTRACT

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’->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

BS and WS 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 were

dependent on nucleoside triphosphate hydrolysis, and required 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.

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INTRODUCTION

Despite the obvious importance of genetic stability, the mammalian

genome has an abundance of sequences that are potentially destabilizing.

Elements which 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 (9,10)). 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) 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),


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and the Xenopus homologue, FFA-1, is required for the formation of replication

foci (23). In addition, recent work suggests a role for the WRN helicase in

telomere maintenance (24-26). 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.

While 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


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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 (48) (reviewed in (49,50), 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 four 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


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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

Proteins Recombinant hexa-histidine tagged human WRN protein was

overexpressed in Sf9 insect cells and purified as described elsewhere (20).

Recombinant hexa-histidine tagged human BLM protein was overexpressed in S.

cerevisiae and purified as previously described (61). UvrD (DNA helicase II) was

kindly provided by Dr. Steven Matson (University of North Carolina at 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 methyl cytosines. This modification

stabilizes the triplexes at physiological pH (62). The 28-mer oligonucleotide 5’-

TCCCAGTCACGACGTTGTAAAACGACGG-3’ was from Gibco-BRL. M13mp18

ss DNA was from New England Biolabs. Ribonucleotides, deoxyribonucleotides,

and ATPγS were from Boehringer Mannheim.

Triplex substrates The plasmid psupF5 contains a duplex sequence that serves

as a target for TC30 and related oligonucleotides. Cleavage of the plasmid with


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NdeI released fragments of 4 and 0.6 kb. The triplex site lies 1800 bases from

one end of the large fragment. Triplexes were prepared by incubation of 3

pmoles of 5’ 32P-labeled oligonucleotide (based on the molecular weights of the

reaction components) overnight at room temperature with 6 pmoles 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 determined in a Perkin Elmer

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 oligos in the buffer described above. The

complexes were then separated from free oligo by ethanol precipitation from 2.5


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M ammonium acetate. They were suspended in 50 mM NaCl, 10 mM Tris-HCl,

pH 7.0, 10 mM MgCl2, 1 mM DTT, 0.1 mM spermine, 100 µg/ml BSA and

digested with Xba I and Pst I 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

32P-labeled TC30. The annealing mixture was incubated at 90 oC 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 non-complementary 5’ tail. The labeled

annealed DNA duplexes were electrophoresed on a 12% polyacrylamide (19:1,

acrylamide: bisacrylamide) 1X Tris-borate, EDTA gel and the duplex fragment

was excised. The DNA fragment was purified using the QIAEX II polyacrylamide

gel extraction kit (Quiagen) and eluted with 10 mM Tris-HCl (pH 8.0). The 28-bp

M13mp18 partial duplex substrate was constructed with a 28-mer

complementary to positions 6296 to 6323 in M13mp18. The 28 bp M13mp18

partial duplex substrates were constructed as previously described (63) with the

following modifications. The 28-mer oligonucleotides were labeled at their 5’

ends using T4 polynucleotide kinase and [γ32P]ATP and annealed to M13mp18


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ssDNA 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 approximately 15 fmoles. The amount of the M13 partial

duplex molecules was 4 fmoles. Reactions were initiated by the addition of WRN

or BLM protein and incubated at 24 oC for 30 min. At the end of the incubation, a

10 µl aliquot of loading buffer (40% glycerol-0.9% sodium dodecyl sulfate (SDS)-

0.1% bromophenol 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 oC. 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 x 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.


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Helicase data shown are representative of at least three independent

experiments.

RESULTS

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 activity of WRN and

BLM enzymes using M13mp18 ssDNA as the DNA effector were determined to

be 219 + 14 min-1 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, while 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,


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eliminating the possibility that triplex unwinding was due to the disruption of the

underlying duplex.

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, while 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 oligos. 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), (our unpublished results). We asked if the helicases would

be active with DNA triplexes with a 3’, or 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.

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


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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 ATPγS 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 effect 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


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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).

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 ATPγS. 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.

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


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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 which 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).

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 6A, B).

DISCUSSION

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


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(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 (67,70-72) (66),

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

suggest 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 (73,74) (75), (76,77), (66,78,79), (80)((81), (82). A possible linkage

between helicase structure and substrate preference has been proposed in a

recent study which 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


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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 non-forked 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 he 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 have observed activity by both enzymes on duplex substrates which do

not have forks (16,20,21,84) (35,38). 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


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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, of 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 (Brosh et al., in preparation). 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


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triplex forming potential are widely distributed in the human genome (1,86,87)

(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.

FIGURE LEGENDS

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) oligo on the supF5 plasmid

with the triplex target. These complexes, as well as the supF5 plasmid without

an oligo (lane 4) or the supF12 plasmid, which lacks the TC30 triplex site, with


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the 3’ tailed oligo (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 ug of

each sample was loaded on each lane. A HindIII digest of phage λ DNA was run

as a molecular weight marker.

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 (x), was incubated with the indicated concentrations of

WRN protein under the standard helicase conditions described in Materials and

Methods. Incubation was at 24oC for 30 minutes. The inset shows the gel

pattern with the 3’ tailed substrate. Heat denatured control (filled triangle).

Fig. 3. Activity of the WRN and BLM helicases on a triplex with a 6 base pair

extension. Heat denatured substrate (filled triangle).

Fig. 4. Activity of the WRN and BLM enzymes with a triplex with no duplex

extension. A. WRN helicase; B. BLM helicase.

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 in Materials and Methods. Incubation

was at 24oC for 30 minutes. Heat denatured control (filled triangle).

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


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indicated concentrations of WRN (A) or BLM (B) enzyme. Heat denatured

control (filled triangle).

Acknowledgements

We thank Dr. Nitin Puri for oligonucleotide synthesis and analysis of the thermal

stability of the triplex and duplex substrates.

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Fig. 1B

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Fig. 2A


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Fig. 4A


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Fig. 4B

BLM (nM)


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WRN (nM) 0 0.9 1.8 3.5 7 14 21 28 ATPγS

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and Michael M. SeidmanRobert M. Brosh ., Jr, Alokes Majumdar, Shital Desai, Ian D. Hickson, Vilhelm A. Bohr

Unwinding of a DNA triple helix by the werner and bloom syndrome helicase

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UNWINDING OF A DNA TRIPLE HELIX BY THE WERNER AND … · ABSTRACT Bloom Syndrome and Werner...

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