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J. Biol. Chem., Vol. 278, Issue 26, 23487-23496, June 27, 2003
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From the
Laboratory of Molecular Gerontology, NIA,
National Institutes of Health, Baltimore, Maryland 21224, the
¶Radiation Oncology Research Laboratory,
Department of Radiation Oncology, University of Maryland, Baltimore, Maryland
21201, and the Department of Biochemistry,
School of Life Sciences, University of Hyderabad, Hyderabad 500 046
India
Received for publication, December 16, 2002 , and in revised form, March 26, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These studies have enabled researchers to speculate about
pathways of DNA metabolism in which WRN might participate; however, the
precise functions of the WRN gene product in vivo are not
well understood.
Recently we reported a novel interaction of WRN protein with the human 5'-flap endonuclease/5' to 3'-exonuclease (FEN-1) (24), a DNA structure-specific nuclease implicated in DNA replication, recombination, and repair (25). WRN protein stimulates FEN-1 cleavage activity by a physical interaction with a COOH-terminal domain of the WRN protein (24). Another member of the RAD2 family to which FEN-1 belongs is human exonuclease 1 (EXO-1) (26). Like FEN-1, EXO-1 is a structure-specific endonuclease as well as an exonuclease (27, 28). EXO-1 has been implicated in a number of DNA metabolic pathways including mismatch correction, mitotic and meiotic recombination, and double strand break repair (29–34). A role for EXO-1 in Okazaki fragment processing during DNA replication has been suggested based on its structure-specific endonuclease and RNase H activities that are similar to those of FEN-1 (30). Functional overlap between EXO-1 and FEN-1 (Rad27 in Saccharomyces cerevisiae) has been proposed from observations in yeast that exoI: rad27 double mutants are inviable (35) and that overexpression of yeast EXO-1 or human EXO-1 complements cellular phenotypes of rad27 mutants (30, 35, 36).
The evident sequence homology and similar biochemical activities of EXO-1 and FEN-1, as well as the potential functional redundancy of the two nucleases, suggested to us that WRN might also interact functionally with EXO-1. We have found this to be the case. Evidence is presented that WRN interacts physically with human EXO-1 and stimulates the endonucleolytic and exonucleolytic cleavage activities of EXO-1. The functional interaction is independent of WRN catalytic function and mediated by a COOH-terminal region of WRN which also interacts with FEN-1. Thus, WRN modulates the cleavage activities of both human EXO-1 and FEN-1 by a direct protein interaction, suggesting that either structure-specific nuclease may act together with WRN during DNA replication, recombination, or repair. The physical and functional interaction of WRN with these human nucleases is likely to be important for the cellular role of WRN in the maintenance of genome integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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WRN-EXO-1 Coimmunoprecipitation Experiments—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. Nuclear extract was prepared as described previously (39). For coimmunoprecipitation, HeLa nuclear extract (1.5 mg of protein) was incubated with goat anti-WRN polyclonal antibody (1:40; Santa Cruz Biotechnology) in buffer D (50 mM HEPES pH 7.5, 100 mM KCl, 10% glycerol) for 4 h at 4 °C and tumbled with 20 µl of protein G-agarose (Roche Applied Science) at 4 °C overnight. Beads were washed three times with buffer D supplemented with 0.1% Tween 20. Proteins were eluted by boiling treatment in SDS sample buffer, resolved on 8% polyacrylamide Tris-glycine SDS gels, and transferred to PVDF membranes (Amersham Biosciences). The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 and probed using rabbit polyclonal antibody against a recombinant human EXO-1 nuclease domain fragment characterized previously (27) (1:5,000; courtesy of Dr. D. M. Wilson III (NIA, NIH)) followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) or probed for WRN using mouse anti-WRN monoclonal antibody (1:250; BD Pharmingen) followed by goat-anti-mouse IgG-horseradish peroxidase (Vector Laboratories). EXO-1 or WRN on immunoblot was detected using ECL Plus (Amersham Biosciences).
Coimmunoprecipitation of purified WRN and EXO-1 was performed in the presence of binding buffer (50 mM Tris, pH 8.0, 10% glycerol, 100 mM NaCl, 0.01% Nonidet P-40) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 5 µg/ml leupeptin). In a 100-µl reaction volume, 500 ng of purified WRN and 500 ng of EXO-1 were incubated with goat anti-WRN polyclonal antibody (1:40) in binding buffer for 4 h at 4 °C. The protein complex was adsorbed on to protein G-agarose beads by incubating the mixture overnight at 4 °C with gentle rotation. The beads were washed three times with binding buffer, eluted by boiling with SDS sample buffer, and resolved on 8–16% gradient SDS-polyacrylamide Tris-glycine gels. After transferring the proteins to PVDF membranes, the membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 and probed with either anti-EXO-1 or anti-WRN antibodies as described above.
Far Western Blotting—Far Western blotting was conducted as described previously (40). Briefly, 0.2–1.0 µg of each protein was electrophoresed on 8–16% SDS-polyacrylamide gels, transferred to PVDF membranes, and processed as described previously (40) with the exception that specified blocked membranes were incubated in the presence of 0.5 µg/ml EXO-1. Membranes were washed, and conventional Western analysis was then performed to detect the presence of EXO-1 using rabbit anti-EXO-1 polyclonal antibody (described above) (1:5,000). Goat anti-rabbit IgG-horseradish peroxidase conjugate was used as secondary antibody at a 1:10,000 dilution and detected using ECL Plus.
Oligonucleotide Substrates—PAGE-purified oligonucleotides
(Midland Certified Reagent Co.) (Table
I) were used for preparation of substrates. Substrates were
prepared as described previously
(41). Briefly, 10 pmol of the
appropriate flap oligonucleotide was 5'-radiolabeled with
[
-32P]ATP and T4 polynucleotide kinase (New England Biolabs)
using the manufacturer's protocol. Unincorporated nucleotide was removed using
a Sephadex G-25 spin column (Amersham Biosciences). The radiolabeled
oligonucleotide was annealed to 25 pmol of the appropriate template
oligonucleotide by heating at 95 °C, then cooling down from 70 to 24
°C. If necessary, 50 pmol of an upstream oligonucleotide was then annealed
to the duplex substrate by heating at 37 °C for 1 h and slowly cooling to
24 °C. Two nicked duplex substrates were prepared: 1) nicked duplex A
using FLAP00A, TSTEM, and U25; and 2) nicked duplex B using FLAP00B, TSTEM2,
and U21. The monodeoxynucleotide (nt) flap was prepared with FLAP01, TSTEM,
and U25. For the monoribodeoxynucleotide flap, FLAP01RNA, TSTEM, and U25 were
used. The 10-nt flap was created with FLAP10, TSTEM2, and U21. The blunt ended
duplex substrate was made using TSTEM-COMP and TSTEM.
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For the nicked duplex substrate with a 3'-end label on the upstream
primer, 10 pmol of U24 was annealed to 25 pmol of TSTEM and end labeled with
[
-32P]dCTP and Klenow fragment (New England Biolabs) at 25
°C for 20 min followed by an additional incubation for 20 min at 25 °C
with 50 µM unlabeled dCTP. Unincorporated nucleotide was removed
by passing over two Sephadex G-25 columns. Klenow was then heat inactivated at
95 °C followed by slow cooling to permit the oligonucleotides to reanneal.
50 pmol of FLAP00 was then added and annealed by heating at 37 °C for 1 h
followed by slow cooling.
EXO-1 Incision Assay—20-µl reactions contained 0.5 nM DNA substrate (unless otherwise noted) and the indicated concentrations of WRN and/or EXO-1 in 30 mM HEPES pH 7.6, 5% glycerol, 40 mM KCl, 0.1 mg/ml bovine serum albumin, and 8 mM MgCl2. WRN was mixed with the substrate on ice prior to the addition of EXO-1. Reactions were incubated at 37 °C for 15 min (unless indicated otherwise), terminated with the addition of 10 µl of formamide dye (80% formamide (v/v), 0.1% bromphenol blue, and 0.1% xylene cyanol), and heated to 95 °C for 5 min. Products were resolved on 20% polyacrylamide, 7 M urea denaturing gels. A PhosphorImager was used for detection, and the ImageQuant software (Molecular Dynamics) was used for quantitation of the reaction products. The percent incision was calculated as described previously (24).
WRN Helicase Assay—20-µl reactions contained 0.5 nM 10-nt 5'-flap DNA substrate (except where indicated) and the indicated concentrations of WRN and/or EXO-1 in the same reaction buffer as used for EXO-1 incision assays except for the additional presence of 2 mM ATP. Reactions were incubated at 37 °C for 15 min, terminated with the addition of 20 µl of helicase stop solution (50 mM EDTA, 40% glycerol, 0.1% bromphenol blue, 0.1% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide with the same sequence as the labeled strand. 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 the ImageQuant software. The percent helicase substrate unwound was calculated as described previously (42).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore the specificity and nature of the suggested WRN-EXO-1 interaction, coimmunoprecipitation experiments were performed with the purified recombinant proteins. Using an antibody directed against WRN, both WRN and EXO-1 were immunoprecipitated (Fig. 1, B and C, lane 5). WRN and EXO-1 were also coimmunoprecipitated in the presence of ethidium bromide confirming that the protein interaction is not mediated through DNA (Fig. 1, B and C, lane 6). Approximately 20% of the input EXO-1 protein was precipitated by the WRN antibody. The purified proteins failed to be immunoprecipitated by goat preimmune serum (Fig. 1, B and C, lane 4) or in the absence of antibody (Fig. 1, B and C, lane 2). In control experiments, EXO-1 failed to be immunoprecipitated by anti-WRN antibody when WRN protein was omitted from the binding mixture (Fig. 1, B and C, lane 3), attesting to the specificity of the WRN antibody used for the coimmunoprecipitation studies. These results indicate a direct physical interaction between WRN and EXO-1 and support the evidence that WRN and EXO-1 interact with each other in vivo.
We next sought to determine whether the COOH-terminal region of WRN responsible for the physical and functional interaction with FEN-1 also interacted physically with EXO-1. To test for this interaction, we performed far Western blotting experiments. For these studies, we tested the ability of full-length WRN protein, a COOH-terminal fragment of WRN (C-WRN940–1432), and an NH2-terminal fragment of WRN (N-WRN1–368) to bind EXO-1. These proteins are shown in a Coomassie-stained SDS-polyacrylamide gel in Fig. 2A. The proteins were resolved on SDS-polyacrylamide gels and transferred to PVDF membranes that were subsequently blocked, incubated in buffer alone or buffer containing EXO-1, and washed extensively. The blots were then probed with an antibody against EXO-1. As shown in Fig. 2B, EXO-1 bound to full-length WRN and the COOH-terminal fragment of WRN (lanes 1 and 2) but did not bind to the NH2-terminal fragment of WRN (lane 3). In the control experiment, when a similar membrane was incubated with buffer lacking EXO-1 and probed with anti-EXO-1 antibody, no bands corresponding to the position of full-length WRN, COOH-terminal or NH2-terminal WRN were detected (Fig. 2C, lanes 1, 2, and 3). In control experiments, two faint bands (fast and slow migrating) were detected in lane 4 containing bovine serum albumin, but this signal was caused by nonspecific reactivity of the EXO-1 antibody, as these bands were also detected on membranes that were incubated in the presence or absence of EXO-1 protein. These results together with the immunoprecipitation experiments demonstrate that WRN and EXO-1 interact physically and that the COOH-terminal region of the WRN protein alone is capable of binding to EXO-1.
WRN Stimulates EXO-1 Cleavage of a 1-Nucleotide 5'-Flap
Substrate—The physical interaction between WRN and EXO-1 and the
ability of WRN to stimulate FEN-1 cleavage by a protein interaction
(24) suggested that WRN might
stimulate the incision activities of human EXO-1. Under the same reaction
conditions, a 1-nt 5'-flap substrate was susceptible to EXO-1 cleavage
that generated the 1-nt product (Fig.
3A, lane 2), whereas FEN-1 cleavage of the same
substrate resulted primarily in the 2 nt product, and to a lesser extent the
1-nt product (Fig. 3A,
lane 6), consistent with previous observations
(24). In the presence of WRN
(4 nM), both EXO-1 and FEN-1 cleavage reactions were stimulated to
yield the same respective cleavage products
(Fig. 3A, lanes
3 and 7). Using a limiting concentration of EXO-1 (0.125
nM), the cleavage reaction was stimulated 8-fold by 8 nM
WRN (Fig. 3B,
lanes 5 and 6, and 3C). Using a 2-fold higher
concentration of EXO-1 (0.25 nM), 10% of the DNA substrate was
cleaved (Fig. 3B,
lane 8, and 3C). At this EXO-1 level, WRN stimulated EXO-1
cleavage to 43% of the substrate incised
(Fig. 3B, lane
9, and 3C). In comparison, 0.25 nM EXO-1 did not
appreciably incise the 5'-32P end-labeled blunt duplex 44-bp
substrate, and the presence of 8 nM WRN resulted in only a small
stimulation of incision activity (
1.5%) using 0.25 nM EXO-1;
however, at a higher concentration of EXO-1 (1 nM), the blunt
duplex DNA substrate was degraded from its 5'-end, and the EXO-1
reaction was stimulated by WRN in a dose-dependent manner (data not shown).
These results indicate that EXO-1 incision at the internally positioned 1-nt
flap was primarily responsible for the removal of the unpaired nucleotide
rather than 5' to 3'-exonuclease digestion from the end of the
blunt duplex substrate. WRN stimulation of EXO-1 cleavage of the 1-nt flap
substrate was also observed in reactions containing 0.5 nM EXO-1
(Fig. 3B, lanes
10 and 11), although the level of stimulation was not as great
because a plateau of incision activity (55%) was approached in the reactions
containing EXO-1 and WRN (Fig.
3C). 8 nM WRN alone did not catalyze
significant cleavage of the 1-nt flap DNA substrate
(Fig. 3B, lane
12), consistent with previous observations
(24). We also observed a
significant stimulation of EXO-1 cleavage of a single ribonucleotide
5'-flap substrate in the presence of WRN using a limiting amount of
EXO-1 (0.125
nM).3
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We subsequently studied EXO-1 cleavage as a function of WRN concentration.
A limiting amount of EXO-1 (0.125 nM) was used such that cleavage
of the 1-nt flap substrate was very low (3%)
(Fig. 3D). A 3-fold
stimulation of EXO-1 cleavage was detected at a WRN concentration of 2
nM (Fig.
3D). In the presence of 4 nM WRN, EXO-1
cleavage increased to 22% incision (Fig.
3D), a 7-fold stimulation of EXO-1 cleavage in the 15-min
incubation. At 8 nM WRN, product formation began to plateau at
30% (Fig.
3D).
Kinetic analysis of the EXO-1 cleavage reaction on the 1-nt 5'-flap
DNA substrate demonstrated that the presence of WRN profoundly affected the
rate of EXO-1 incision (Fig.
4). In these studies, we used a concentration of WRN (8
nM) which was previously determined to achieve maximal stimulation
of EXO-1 cleavage (Fig.
3D). The concentration of EXO-1 (0.125 nM)
used resulted in a low, but reproducibly detectable incision of 2% of the
0.5 nM DNA substrate in a 15-min reaction in the absence of WRN
(Fig. 3D). Stimulation
of EXO-1 incision by WRN was detected at time points as short as 1–2 min
(Fig. 4). Up to 9 min, EXO-1
cleavage in the absence of WRN was 1%; however, in the presence of WRN,
EXO-1 incised 19% of the DNA substrate at the 9 min time point. Regression
analysis of the linear regions of the slopes yielded reaction rates of 25.9
and 0.7 pmol of product/min for the WRN + EXO-1 and EXO-1 reactions,
respectively. This difference represented a 37-fold increase in the rate of
EXO-1 cleavage when WRN is present. At 15 min, the EXO-1 cleavage reaction
conducted in the presence of WRN achieved a plateau of 28% substrate
incised. In contrast, EXO-1 alone cleaved only 1.5% of the substrate by the
end of 15 min. These results demonstrate conclusively that WRN stimulates the
rate of EXO-1 cleavage.
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Catalytic Activities of WRN Are Not Required for Stimulation of EXO-1
Incision—Our previous work demonstrated that a protein domain of
WRN, devoid of catalytic activities and which interacts physically with FEN-1,
mediated the functional interaction between WRN and FEN-1
(24). However, it was
conceivable that WRN might affect the cleavage activity of EXO-1 by a
mechanism distinct from that of the WRN-FEN-1 interaction. The three catalytic
activities of WRN (ATPase, helicase, exonuclease) might influence the
functional interaction with EXO-1. To address this possibility, we tested the
effects of full-length recombinant WRN proteins with site-directed mutations
in the active sites of its catalytic domains on EXO-1 cleavage. WRN-K577M
mutant protein, devoid of ATPase or helicase activity
(18,
43), was capable of
stimulating EXO-1 cleavage of the 1-nt flap DNA substrate similarly to
wild-type WRN resulting in 30% incision
(Fig. 5A, lanes
3 and 6) compared with 2% incision in reactions containing only
EXO-1 (Fig. 5A,
lane 2). WRN-E84A, defective in exonuclease activity
(21,
38), was able to stimulate
EXO-1 cleavage similarly to the wild-type or ATPase mutant WRN proteins
(Fig. 5A, lane
4). The WRN-K577M mutant protein preparation contained a small level of
nuclease activity which digested 2% of the substrate
(Fig. 5A, lane
10), whereas the WRN-E84A was free of nuclease activity on the
5'-flap substrate (Fig.
5A, lane 8). These results indicate that WRN
exonuclease or helicase/ATPase activities are not required for the stimulation
of EXO-1 nuclease activity by WRN. We also tested a truncated recombinant WRN
protein (residues 1–368) which lacks the ATPase/helicase and
COOH-terminal region of full-length WRN protein but retains the exonuclease
domain and possesses intrinsic 3' to 5'-exonuclease activity
(44). The
N-WRN1–368 protein failed to stimulate EXO-1 incision
(Fig. 5A, lane
5), suggesting that the functional interaction between WRN and EXO-1 is
specific and may be mediated by the COOH-terminal region of WRN.
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The ability of the catalytically defective mutant WRN proteins to stimulate EXO-1 cleavage suggested that the functional interaction is mediated by a direct protein interaction. We demonstrated previously that a COOH-terminal fragment (sequence 940–1432) of WRN which physically interacts with FEN-1 is capable of stimulating FEN-1 cleavage of a 1-nt 5'-flap substrate (24). To determine whether this domain of WRN is sufficient to stimulate EXO-1 cleavage, we tested directly the effect of purified C-WRN940–1432 on EXO-1 cleavage of the 1-nt 5'-flap substrate (Fig. 5B). 0.25 nM EXO-1 incised 1, 4, and 13% of the 1-nt 5'-flap substrate in the presence of 0, 4, and 8 nM C-WRN940–1432, respectively (Fig. 5B, lanes 2, 3, and 4). No incision was detected in reactions containing only C-WRN940–1432 (Fig. 5B, lanes 5 and 6). The ability of the highly purified C-WRN940–1432 fragment to stimulate EXO-1 cleavage indicates that the protein domain responsible for stimulating EXO-1 cleavage resides within the COOH terminus of WRN, a region of the protein which interacts physically with EXO-1 and was shown to mediate the functional interaction with another Rad2 family member, human FEN-1 (24).
WRN Stimulates EXO-1 Incision of a Nicked Duplex DNA Substrate—It was observed recently that EXO-1 is required in a reconstituted mismatch repair reaction using human nuclear extracts with a DNA substrate where mismatch provoked incision is directed by a strand break located either 5' or 3' to the mismatch (45). A reported mismatch repair deficiency in WS fibroblast cell lines may be attributed to either a direct role of WRN in the repair pathway or the consequence of a secondary mutation in a mismatch repair gene which results in a loss of mismatch repair activity (46). We therefore set out to test the effect of WRN on EXO-1 cleavage activity of a proposed DNA mismatch repair intermediate, a nicked duplex DNA substrate. Two nicked duplex substrates of different sequences were tested with the 5'-32P label residing on the 19-nt and 21-nt downstream primers of nicked duplex substrates A and B, respectively. The nicked duplex substrates were verified for their integrity by performing ligation reactions using T4 DNA ligase. Approximately 95% of nicked duplex substrates was converted to a ligated product of the upstream and downstream primers, providing evidence that they are indeed nicked duplex molecules (data not shown). 8 nM WRN stimulated 0.25 nM EXO-1 to remove the 5'-32P-labeled nt of the downstream primer of either nicked duplex substrate A or B (Fig. 6A, lanes 3 and 7). In control reactions, 8 nM WRN alone did not catalyze any significant incision of the nicked duplex substrate, whereas higher concentrations of EXO-1 cleaved nicked duplex substrate A or substrate B (data not shown).
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A quantitative analysis of EXO-1 cleavage of nicked duplex substrate A in
the presence of 8 nM WRN as a function of EXO-1 concentration is
shown in Fig. 6B. 0.5
nM EXO-1 incised 12% of nicked duplex substrate A. Using a limiting
concentration of EXO-1 (0.125 nM), the cleavage reaction was
stimulated 8-fold by 8 nM WRN, resulting in 26% incision
(Fig. 6B). Using a
2-fold higher concentration of EXO-1 (0.25 nM), 5% of the nicked
DNA substrate was incised (Fig.
6B). At this EXO-1 level, WRN stimulated EXO-1 incision
to 42% of the substrate acted upon. Similar results were obtained using 0.5
nM EXO-1, although the degree of stimulation (6-fold) was not
as great because the incision level began to plateau
(Fig. 6B).
We next examined EXO-1 cleavage of the nicked duplex DNA substrate A as a
function of WRN concentration. A limiting concentration of EXO-1 (0.125
nM) was used such that incision of the nicked duplex was very low
(1.5%) (Fig. 6C).
A statistically significant stimulation of EXO-1 cleavage was observed at a
WRN concentration of 2 nM (Fig.
6C). At this WRN level, EXO-1 incised 20% of the
nicked DNA substrate (Fig.
6C). Further increase of the WRN concentration resulted
in greater amount of EXO-1 digestion of the nicked duplex. At 20 nM
WRN, EXO-1 cleaved 33% of the substrate, a 15-fold increase compared with
reactions containing only EXO-1 (Fig.
6C).
It was speculated that EXO-1 might play a role in 3'-hetero-duplex repair by having a regulatory/structural role in 3'-excision complex assembly or by a cryptic 3' to 5' hydrolytic activity (45). We tested a nicked duplex substrate with a 3'-end label on the upstream primer and found that 0.125 nM EXO-1 in the absence or presence of 20 nM WRN did not incise the 3'-label under conditions where the 5'-end label of the downstream primer was catalytically removed (data not shown), suggesting that WRN does not reverse the 5' to 3' polarity of the exonuclease reaction catalyzed by EXO-1 at the site of a nick in a DNA duplex substrate. Furthermore, the results with the nicked duplex substrate containing a 3'-end label on the upstream primer suggest that the incision detected on the nicked duplex with the 5'-label on the downstream primer is caused by WRN stimulation of EXO-1 cleavage at the site of the nick. We conclude that the 5' to 3'-exonuclease activity of EXO-1 at a nick on duplex DNA is stimulated by WRN, and EXO-1 does not exhibit a 3' to 5'-exonuclease activity on these substrates under these conditions.
WRN Stimulates the Endonucleolytic Cleavage Activity of
EXO-1—Recent biochemical evidence has demonstrated that full-length
S. cerevisiae EXO-1 as well as a protein fragment of human EXO-1
which harbors the conserved nuclease motifs of Rad2 family members (FEN-1,
XPG, EXO-1) display 5'-flap endonuclease activity
(27,
28). It has been suggested
that the structure-specific endonuclease activity of EXO-1 may be important
for biological function in DNA metabolic pathways, most notably Okazaki
fragment maturation during DNA replication. We therefore examined the effect
of WRN on the flap endonuclease activity of EXO-1 to determine whether WRN
might also stimulate structure-specific incision in addition to its positive
effect on the 5' to 3'-exonuclease activity of EXO-1. The results
shown in Fig. 7 demonstrate
that this is the case. 1 nM EXO-1 endonucleolytically incised
4% of a 10-nt 5'-flap substrate. The 10-nt incision product was the
same as that shown previously to be endonucleolytically incised by a purified
protein fragment of EXO-1 (27)
(Fig. 7A, lane
2). Thus full-length human EXO-1 has intrinsic flap endonuclease
activity. We also observed some exonuclease activity on the 5'-flap as
detected by the appearance of mononucleotide
(Fig. 7A, lane
2), a result consistent with that observed when the purified nuclease
domain fragment of EXO-1 was incubated with the same flap substrate
(27). At low concentrations of
WRN (0.5 and 1 nM), a small stimulation of EXO-1 incision
(1.5-fold) was observed (Fig.
7A, lanes 4 and 5). Using 2
nM WRN, EXO-1 cleaved 9% of the 10-nt 5'-flap DNA substrate
(Fig. 7A, lane
6). EXO-1 cleavage was elevated further in the presence of 4 or 8
nM WRN, yielding 21 and 31% incision, respectively
(Fig. 7A, lanes
7 and 8). Importantly, in control reactions 8 nM WRN
alone did not incise the 10-nt 5'-flap structure
(Fig. 7A, lane
9), as reported previously
(42). At high concentrations
of WRN (8 nM) (lane 9), a very low level of
5'-nuclease activity could be detected which was also evident in
reactions containing exonuclease-defective WRN (data not shown), suggesting
that a minor contaminant in the WRN protein preparation is responsible for
this activity. Thus WRN is competent to stimulate the EXO-1 endonucleolytic
cleavage reaction, as great as 8-fold, on the 10-nt 5'-flap substrate.
We also observed stimulation of the EXO-1 5' to 3'-exonuclease
activity on the 5'-ssDNA flap by WRN, resulting in 5 and 17% incision at
WRN concentrations of 4 and 8 nM, respectively
(Fig. 7A, lanes
7 and 8). The ability of WRN to stimulate 5' to
3'-exonuclease activity of EXO-1 on the 5'-ssDNA flap
oligonucleotide is consistent with our observation that EXO-1 incision of
5'-32P-labeled ssDNA oligonucleotide is also stimulated by
WRN (data not shown).
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We next tested the purified COOH-terminal protein fragment of WRN
(C-WRN940–1432), devoid of the helicase and exonuclease
domains, for its ability to stimulate EXO-1 cleavage of the 10-nt
5'-flap substrate. Using 1 nM EXO-1, the cleavage reaction
was stimulated 15-fold by 4 nM C-WRN940–1432
(Fig. 7B, lane
3). Slightly higher EXO-1 cleavage (37%) was observed using 8
nM C-WRN940–1432
(Fig. 7B, lane
4). The 10-nt product from reactions containing
C-WRN940–1432 and EXO-1 were also detected from reactions
containing EXO-1 + full-length WRN (Fig.
7A) or higher concentrations of EXO-1 (data not shown),
indicating that the cleavage site specificity of EXO-1 is not altered by the
WRN COOH-terminal protein fragment. Purified N-WRN protein fragment failed to
stimulate endonucleolytic or exonucleolytic incision by EXO-1 (data not
shown), adding further evidence that the functional interaction between WRN
and EXO-1 is the result of a protein interaction mediated by the COOH-terminal
region of WRN. The levels of stimulation of EXO-1 endonucleolytic cleavage by
C-WRN940–1432 were comparable with those observed with the
full-length WRN protein (Fig.
7B, lanes 7 and 8). These results
indicate that the protein domain of WRN which physically binds to EXO-1 is
capable of stimulating EXO-1 cleavage of the 10-nt 5'-flap
substrate.
WRN Stimulates EXO-1 Cleavage as Effectively as FEN-1
Cleavage—Because WRN can stimulate either EXO-1 or FEN-1
endonucleolytic incision of 5'-flap substrates, we wanted to compare the
-fold stimulation by WRN of the two human Rad2 nucleases. Using the 10-nt
5'-flap substrate, WRN was tested for its effect on EXO-1 or FEN-1
cleavage under the identical reaction conditions. The concentrations of EXO-1
(0.5 nM) and FEN-1 (0.06 nM) were adjusted to achieve a
similar level of endonucleolytic cleavage (5%) of the 5'-flap
substrate (Fig. 7C).
In the presence of 8 nM WRN, EXO-1 cleavage was increased 6-fold,
and FEN-1 cleavage was increased 7-fold
(Fig. 7C). These
results indicate that the stimulatory effect of WRN on flap cleavage by the
two human endonucleases is very similar.
Both RPA and PCNA stimulate FEN-1 cleavage (41, 47), raising the possibility that these human nuclear proteins might stimulate EXO-1 cleavage; however, there is no previous report of the effect of RPA or PCNA on human EXO-1 cleavage. At molar concentrations of PCNA homotrimer or RPA heterotrimer equal to WRN monomer (8 nM) that displayed a 16-fold stimulation of EXO-1 endonucleolytic cleavage of the 10-nt flap substrate, we did not detect any significant stimulation of EXO-1 cleavage (endonucleolytic or exonucleolytic) by either PCNA or RPA (data not shown).
WRN Stimulates EXO-1 Endonucleolytic Cleavage under Conditions
Favorable for Helicase-catalyzed DNA Unwinding—Recently, we
reported that WRN helicase efficiently unwinds a 10-nt 5'-flap
substrate, releasing the 5'-flap oligonucleotide, leaving the duplex
species containing the upstream oligonucleotide intact
(42). To determine the outcome
of 5'-flap processing by EXO-1 on a substrate that is also unwound by
WRN, we examined both DNA unwinding (release of the 5'-flap
oligonucleotide) and EXO-1 cleavage of the 5'-flap oligonucleotide in
the presence of ATP, which fuels the WRN helicase reaction. The products were
analyzed on nondenaturing polyacrylamide gels, and the results of a typical
experiment are shown in Fig. 8.
4 or 8 nM WRN stimulated cleavage of the flap substrate by 0.5
nM EXO-1 10-fold and 6-fold, respectively
(Fig. 8, lanes 3 and
4) as detected by the increased amount of the 10-nt incision product
that comigrated with the radiolabeled 10-nt marker indicated on the
left. Under these conditions in the presence of 0.5 nM
EXO-1, 4 or 8 nM WRN unwound 8 and 39% of the flap substrate,
respectively (Fig. 8, lanes
3 and 4). The oligonucleotide released by WRN helicase activity
comigrated with the intact 31-mer (FLAP10) used in the preparation of the
5'-flap substrate. Similar levels of WRN helicase activity (14 and 44%)
were detected in reactions containing only 4 or 8 nM WRN (lanes
5 and 6). When EXO-1 was increased to 2 nM, 7% of the
5'-flap substrate was endonucleolytically incised in the absence of WRN
(lane 7). In the presence of WRN, endonucleolytic incision by EXO-1
was again increased significantly as detected by the appearance of the 10-nt
incision product (lanes 8 and 9). The percent
endonucleolytic incision by 2 nM EXO-1 in the presence of 4 or 8
nM WRN was 41 and 18%, respectively. The reduction in EXO-1 10-nt
cleavage product in the presence of 8 nM WRN compared with 4
nM WRN is accompanied by a greater amount of the intact flap
oligonucleotide released. WRN also stimulates EXO-1 degradation of the
released 5'-ssDNA flap and/or the 5'-ssDNA end of the intact flap
structure as evidenced by comigration of the fast migrating species (lanes
3, 4, 8, and 9) with [32P]AMP. Only 19% of the intact
31-mer was released by 8 nM WRN in the presence of 2 nM
EXO-1 compared with 44% flap oligonucleotide released by WRN alone, indicating
a 2.3-fold reduction in the release of intact flap oligonucleotide. These
results indicate that under conditions in which WRN is active as a helicase
(presence of ATP), WRN can stimulate EXO-1 endonucleolytic incision or unwind
the 5'-flap oligonucleotide.
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DISCUSSION |
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The ability of WRN to stimulate the incision reactions catalyzed by the sequence-related human nucleases EXO-1 and FEN-1 on short (1-nt) and long (10-nt) 5'-ssDNA flap substrates suggests that the functional interaction may proceed by a similar mechanism. In both cases, the stimulation of incision is mediated by a COOH-terminal domain of the WRN protein. Although the WRN interaction site on FEN-1 is not known, it will be of interest to determine whether the site of WRN physical interaction is the same for EXO-1 and FEN-1. A number of similarities exist between EXO-1 and FEN-1, but there are some noticeable differences in their DNA substrate specificities and cleavage products (27, 28). For the structures tested thus far, the DNA substrate specificities of EXO-1 and FEN-1 are not altered by the presence of WRN. WRN stimulates EXO-1 cleavage of ssDNA and blunt duplex dsDNA, whereas FEN-1 acts poorly on these substrates, and WRN does not appreciably stimulate FEN-1 on either of these structures (24). Although WRN is able to stimulate structure-specific endonucleolytic cleavage of 5'-flap structures by EXO-1 and FEN-1, the nuclease reactions display a subtle difference in the products generated. WRN stimulation of EXO-1 yields a 1-nt product from the 1-nt 5'-flap substrate, consistent with the cleavage products obtained from reactions containing higher levels of EXO-1 alone. Thus EXO-1 cleaves primarily at the base of the flap, yielding a nicked duplex intermediate that can be readily sealed by ligase. In contrast, FEN-1 (in the absence or presence of WRN) incises primarily 1 nt into the downstream annealed region, and to a lesser extent at the base of the flap, resulting in primarily a 1-nt gap duplex and a lower amount of nicked duplex, respectively (24, 41). This difference may be important during Okazaki fragment processing of double flap substrates in which the upstream primer has a single unannealed nt, a preferred DNA substrate for FEN-1 cleavage (48). By the preferential FEN-1 incision of the 5'-flap 1 nt into the annealed downstream region, the unannealed (but complementary) nt at the 3'-end of the upstream primer may anneal to form a ligatable nicked DNA duplex substrate. It will be of interest to determine whether human EXO-1 also prefers a double flap structure as a cleavage substrate; alternatively, the cleavage specificity of EXO-1 may be well suited to process 5'-flap structures that lack 3'-flaps, a reaction that may also be important during eukaryotic DNA replication or some other pathway of DNA metabolism.
In which cellular pathways is the functional interaction of WRN and EXO-1 likely to be important? Yeast genetic studies have implicated EXO-1 in spontaneous mitotic and meiotic recombination between direct repeats (29). In one model, the 5' to 3'-exonuclease activity of EXO-1 may act to generate 3'-ssDNA tails that can be used by homologous pairing proteins for recombination. EXO-1 has also been implicated in the processing of complementary double strand ends in a recombination pathway of double strand break repair. More specifically, it was recently proposed that the 5' to 3'-exonuclease activity of EXO-1 partially compensates for a deficiency in the function of the Rad50·Mre11·Xrs2 complex in homologous recombination repair (32, 34). Although a precise role of the WRN protein in a specific recombination pathway remains to be defined, there are several lines of evidence suggesting that a role of WRN in recombination is likely. 1) WS cells display severe genomic instability, elevated homologous recombination (9), and aberrant mitotic recombination (10, 49). 2) the human WRN gene can suppress the increased homologous and illegitimate recombination in the yeast WRN homolog sgs1 (50). 3) WRN helicase can recognize and unwind a number of DNA structures with junctions (42) and catalyzes branch fork migration of Holliday junctions (51), a key DNA intermediate of homologous recombination. The results presented in this report suggest that WRN may function to modulate the cleavage activity of EXO-1 which is thought to be essential to the role of the enzyme in homologous recombination.
EXO-1 has been implicated in mismatch repair by its requirement in mutation avoidance (30) and interactions with mismatch repair proteins (33, 35). Very recently, EXO-1 was demonstrated to be required in mismatch repair in a reconstituted system using extracts from human cells (45). Human EXO-1 was shown to perform mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. Evidence that WRN protein may play a role in mismatch repair was provided by the demonstration that extracts from three WS fibroblastoid cell lines were deficient in repair of base-base and insertion/deletion mismatches (46). However, in the same study, extracts from four WS lymphoblastoid cell lines were all proficient in mismatch repair, suggesting that WRN protein could have a cell type- and/or tissue-specific role in mismatch repair. It was reported subsequently that introduction of human chromosome 8 encoding the WRN gene to WS fibroblast cell lines deficient in mismatch repair resulted in expression of WRN protein but did not restore mismatch repair activity (52). Thus, it is unclear whether or not WRN plays a direct role in mismatch repair. Based on our results that WRN and EXO-1 interact functionally, it is possible that WRN modulates EXO-1 activity in a pathway of mismatch repair. The elevated frequency of spontaneous mutations in the HPRT gene (4) and the pronounced genomic instability of WS cells (2, 3) may reflect at least in part a defect in a mutation avoidance pathway that relies on the mismatch repair machinery, including EXO-1.
It has been suggested that EXO-1 plays a role in Okazaki fragment processing in yeast that is at least partially redundant to FEN-1 (RAD27). On a biochemical level, EXO-1 is able to remove RNA primers from Okazaki fragment model substrates efficiently, suggesting that the enzyme may function in RNA primer removal during lagging strand synthesis (30). The ability of human EXO-1 to suppress the lethality of a rad27 mutant at the restrictive temperature is consistent with this notion (30). WRN stimulation of EXO-1 5'-flap cleavage may serve a function during Okazaki fragment maturation or the processing of other DNA structures at the replication fork. Evidence presented here demonstrates that WRN is able to stimulate EXO-1 cleavage of flap structures with 5'-ssDNA tails up to at least 10 nt, an activity that may be important in a FEN-1 independent pathway of 5'-flap processing (53). Alternatively, EXO-1 may play a role in the removal of the terminal ribonucleotide at the RNA-DNA junction during Okazaki fragment processing in a WRN-stimulated reaction. WRN may serve as a general factor to promote replication intermediate processing through its interaction with structure-specific endonucleases.
Very recently it was reported that EXO-1-dependent ssDNA at telomeres is required for cell cycle arrest in budding yeast ku70 deletion mutants (54). The observation that EXO-1 affects the metabolism of damaged telomeres and checkpoint responses is interesting in light of circumstantial evidence that WRN may also play a role in telomere metabolism. WS fibroblasts that express a transfected human telomerase (hTERT) gene have an extended lifespan and can become immortalized (55), suggesting that the absence of WRN protein confers accelerated cellular senescence in a pathway dependent on telomerase. However, WRN may also function in an alternate telomerase-independent mechanism, as suggested by studies of the yeast WRN homolog Sgs1 (56, 57). Although no direct role of WRN protein in telomere metabolism has been established, it is conceivable that WRN stimulation of EXO-1 nuclease activity in subtelomeric regions is important for the replication and stability of telomeres and/or the activation of the appropriate DNA damage and signal checkpoint pathways to tolerate damaged telomeres.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Laboratory of Molecular Gerontology, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8578; Fax: 410-558-8157; E-mail: BroshR{at}grc.nia.nih.gov.
1 The abbreviations used are: WS, Werner syndrome; dsDNA, double strand DNA;
EXO-1, exonuclease 1; FEN-1, 5'-flap endonuclease/5' to
3'-exonuclease; nt, nucleotide; PCNA, proliferating cell nuclear
antigen; PVDF, polyvinylidene difluoride; RPA, replication protein A; ssDNA,
single strand DNA.
2 L. A. Uzdilla, J. P. Carney, P. Hungspreugs, D. M. Wilson, and T. M.
Wilson, manuscript in preparation.
3 S. Sharma, J. A. Sommers, H. C. Driscoll, L. Uzdilla, T. M. Wilson, and R.
M. Brosh, Jr., unpublished data.
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ACKNOWLEDGMENTS |
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