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Nucleic Acids Research Advance Access originally published online on August 22, 2007
Nucleic Acids Research 2007 35(17):5706-5716; doi:10.1093/nar/gkm500
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Nucleic Acids Research, 2007, Vol. 35, No. 17 5706-5716
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors

Nurten Saydam1, Radhakrishnan Kanagaraj1, Tobias Dietschy1,2,3, Patrick L. Garcia1, Javier Peña-Diaz1, Igor Shevelev2,3, Igor Stagljar2,3 and Pavel Janscak1,*

1Institute of Molecular Cancer Research of the University of Zurich, Switzerland, 2Department of Biochemistry and 3Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada

*To whom correspondence should be addressed. Tel: +41(0)44 635 3470; Fax: +41(0)44 635 3484; Email: pjanscak{at}imcr.unizh.ch

Received April 26, 2007. Revised June 7, 2007. Accepted June 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3'-5' exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutS{alpha}), MSH2/MSH3 (MutSß) and MLH1/PMS2 (MutL{alpha}) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutS{alpha} and MutSß can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3'-single-stranded arm. The stimulatory effect of MutS{alpha} on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutL{alpha} protein known to bind to the MutS {alpha}–heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutS{alpha}, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Werner syndrome (WS) is an autosomal recessive disorder characterized by an early onset of age-related pathologies including graying hair, alopecia, arteriosclerosis, osteoporosis, diabetes mellitus and cancer (1). The gene mutated in WS, WRN, encodes a RecQ-type DNA helicase (2,3). WRN also possesses a 3' - 5' exonuclease activity residing in a separate domain located at the N-terminus of the protein (4,5). At the cellular level, WRN deficiency is associated with defects in DNA replication, homologous recombination (HR) and telomere maintenance (6–9). As a result, cells derived from WS patients display a high degree of genomic instability including elevated levels of chromosomal translocations and deletions (10–13). WS cells are hypersensitive to DNA-damaging agents such as 4-nitroquinoline 1-oxide, topoisomerase inhibitors and DNA cross-linkers, suggesting that WRN is actively involved in DNA repair (14–16). Several lines of evidence implicate WRN in the cellular response to DNA double-strand breaks (DSBs). WRN is rapidly recruited to the sites of ionizing radiation (IR)-induced damage (17). Moreover, it interacts physically and functionally with a number of proteins that are involved in the DSB-repair process including the MRE11-RAD50-NBS1 (MRN) complex (18), the Ku complex (19), RAD52 (20) and DNA-dependent protein kinase (21). However, the precise role for WRN in DNA repair remains to be elucidated.

The DNA mismatch-repair (MMR) system maintains genomic integrity by correcting DNA replication errors and preventing recombination between divergent sequences (22,23). Defects in a subset of MMR genes including MSH2, MSH3, MSH6, MLH1 and PMS2 are associated with hereditary non-polyposis colon cancer, highlighting the crucial role for MMR in genome maintenance (24). In the initiation step of the eukaryotic MMR process, at least three heterodimers, namely MSH2/MSH6 (MutS{alpha}), MSH2/MSH3 (MutSß) and MLH1/PMS2 (MutL{alpha}), are involved (22). MutS{alpha} binds to base-base mismatches and short insertion/deletion loops, while MutSß can recognize only insertion/deletion loops containing up to 16 extra nucleotides in one strand (22). MutL{alpha} possesses an intrinsic endonuclease activity, which is activated upon mismatch recognition and introduces incisions in the discontinuous strand of the heteroduplex DNA, generating entry sites for the 5'-3' exonuclease EXO1 (25).

Sgs1, the yeast ortholog of WRN, also contributes to the suppression of recombination between divergent DNA sequences (26). Heteroduplex rejection during repair of DSBs by the single-strand annealing pathway of HR in yeast requires the mismatch binding and ATPase functions of the Msh2p/Msh6p heterodimer and the helicase activity of Sgs1 (27,28). These findings led to the proposal that MMR proteins act in conjunction with Sgs1 to unwind DNA recombination intermediates containing mismatches (27,28).

Here we demonstrate that WRN directly interacts with MutS{alpha}, MutSß and MutL{alpha} via distinct domains. MutS{alpha} and MutSß are found to stimulate WRN-mediated unwinding of forked DNA duplexes with a 3'-single-stranded (ss) arm. The stimulatory effect of MutS{alpha} on WRN-mediated unwinding is enhanced by a single G/T mismatch located in the duplex ahead of the fork in a manner independent of MutL{alpha}. These data provide biochemical evidence suggesting that the rejection of homeologous recombination by MMR proteins occurs via helicase-mediated unwinding of recombination intermediates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Construction of plasmids
The bacterial expression vectors for the WRN fragments encompassing the amino acid residues 51–449, 949–1432, 500–946, 500–1149, 500–1236, respectively, fused to the C-terminus of glutathione S-transferase (GST) were constructed by PCR amplification of corresponding regions of the WRN cDNA and their insertion in pGEX-2TK (Amersham Biosciences) between the EcoRI and BamHI sites. The complete coding region of WRN was amplified by PCR and cloned in pACT2 (Clontech Palo Alto, CA) via SmaI site to construct a yeast two-hybrid (YTH) vector expressing WRN as a fusion with a Gal4 activation domain. MLH1 cDNA comprised of the codons 500–756 was cloned in a YTH vector pBTM116 (Clontech Palo Alto, CA) between the EcoRI and SalI sites, resulting in a fusion with a LexA DNA binding domain. The pBTM16 derivatives expressing other MLH1 variants as well as the full-length yMlh1 were previously described (29). The Gal4-hMSH2 (pLJR105), LexA-MSH3 and LexA-MSH6 bait plasmids were also described previously (30,31).

Proteins
Recombinant human WRN (3,32), MutS{alpha} (33), MutSß (33) and MutL{alpha} (34) and Escherichia coli MutS (35) were produced and purified as previously described. An antibody against the N-terminal region of WRN encompassing amino acids 1–391 (ISEV-391) was raised in rabbit and purified on an antigen-coupled Sepharose 4A column (Amersham Biosciences). Control IgGs were purified from a rabbit preimmune serum on a 5 ml HiTrap protein G-Sepharose column (Amersham Biosciences).

Cell culture
The following human cell lines were used in this study: HEK 293 embryonic kidney cells and AG11395 SV40-transformed WS fibroblasts (Coriell Institute for Medical Research). The HEK 293 cells were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum (Biochrome AG). The WS cells were maintained in MEM containing 15% fetal calf serum and 2 mM L-glutamine.

Immunoprecipitation assays
Cells were suspended in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol and complete, EDTA-free protease inhibitor cocktail (Roche). After sonication, the suspension was centrifuged at 20 000 g for 30 min at 4°C. Aliquots containing 1.6 mg of protein were incubated overnight at 4°C with purified rabbit polyclonal anti-WRN IgGs (2 µg), which was followed by a 2-h incubation with protein A/G-agarose beads (Santa Cruz) at 4°C. Where required, extracts were treated with 50 U of DNaseI (Roche) for 30 min at 25°C prior to addition of antibody. After extensive washing with the lysis buffer, the immunoprecipitates were subjected to electrophoresis in a 7.5% polyacrylamide–SDS gel followed by western blotting. The blots were probed with mouse monoclonal antibodies against WRN (BD Biosciences, 611169), MSH6 (Pharmingen, clone 44), PMS2 (Pharmingen, clone 16-4), MLH1 (BD Biosciences, 554073), MSH2 (Calbiochem, clone NA 26) and MSH3 (Transduction Laboratories, clone 52). Immune complexes were detected using ECL-plus reagent (Amersham Biosciences), with horse anti-mouse IgG-horseradish peroxidase conjugate (Vector) used as a secondary antibody. In the control experiment, IgGs purified from a preimmune rabbit serum were used instead of the anti-WRN antibody.

GST pull-down assays
GST–WRN fusion proteins were produced in the E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene) using the expression vectors described above. The fusion proteins were bound to glutathione–sepharose beads (Amersham Biosciences) as previously described (36). The beads were incubated with 1 µg of purified MutS{alpha}, MutSß or MutL{alpha} in 400 µl of NET-N 100 buffer [10 mM Tris HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.5% (v/v) NP-40] for 2 h at 4°C. After extensive washing with NET-N 100 buffer, proteins bound to the beads were analyzed by western blotting. Membranes were probed with the monoclonal antibodies described above. MSH3 was detected with a rabbit polyclonal antibody (NTH3) raised against its first 200 amino acids (Eurogentec). In a control experiment, beads were coated with GST protein only.

ELISA-based protein binding assay
Purified recombinant WRN was diluted to a concentration of 20 nM in carbonate buffer [16 mM Na2CO3, 34 mM NaHCO3 (pH 9.6)] and added to wells of a 96-well microtiter plate (50 µl/well). Plates were incubated overnight at 4°C. For control reactions, wells were pre-coated with an equivalent amount of bovine serum albumin (BSA). After aspiration of the samples, the wells were blocked with blocking buffer [phosphate-buffered saline, 0.5% (v/v) Tween-20 and 3% (w/v) BSA] for 2 h at 37°C (200 µl/well). Following blockage, the wells were incubated with increasing concentrations of purified recombinant MutS{alpha}, MutSß and MutL{alpha} proteins for 1 h at 37°C. All samples were supplemented with ethidium bromide (EtBr) at a concentration of 50 µg/ml to prevent DNA-mediated interactions. Wells were washed four times with blocking buffer to eliminate unbound proteins, and incubated with the appropriate primary antibody diluted in blocking buffer (mouse monoclonal anti-MSH2 antibody for MutS{alpha} and MutSß, and mouse monoclonal anti-MLH1 antibody for MutL{alpha}). Plates were incubated for 1 h at 37°C. After four washings with blocking buffer, horseradish peroxidase-conjugated anti-mouse secondary antibody (1:10 000 in blocking buffer) was added and the plates were incubated at 37°C for 30 min. After extensive washing with blocking buffer, the protein complexes were detected using o-Phenylenediamine dichloride (Sigma) dissolved in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.03% hydrogen peroxide (1 mg/ml). The reactions were terminated after 5 min by adding 50 µl of 2 M H2SO4. The plates were scanned in a microplate reader (Molecular Devices) for absorbance at 492 nm. The A492 values, corrected for background signal in the presence of BSA, were plotted as a function of the concentration of appropriate MMR protein using the GraphPad Prism software. To determine an apparent dissociation constant of each complex (Kd), the data points were fitted by the hyperbolic function Y = Bmax*X/(Kd + X) where Bmax is the maximal binding and Kd is the concentration of ligand required to reach half-maximal binding.

YTH assay
YTH analysis was carried out using Saccharomyces cerevisiae strains L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) and Y190 (MAT{alpha}, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4{Delta}, gal80{Delta}, cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-lacZ). The former strain was used for LexA-bait vectors while the latter strain was used for Gal4-bait vectors. Clones carrying the bait and prey plasmids were tested for ß-galactosidase activity using a pellet X-gal (PXG) assay as previously described (37).

Helicase assays
Schemes of DNA substrates as well as the sequences of the constituent oligonucleotides are summarized in Supplementary Table 1. The f11-20 oligonucleotide (50-mer) was labeled at the 5'-end using T4 polynucleotide kinase (NEB) and [{gamma}-32P]ATP (Amersham Biosciences), and annealed with appropriate oligonucleotides under previously described conditions (38). The helicase reaction mixtures (10 µl) contained 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 50 µg/ml of BSA, 2 mM ATP, 1 mM DTT, 1 nM DNA substrate and indicated concentrations of MMR proteins (MutS{alpha}, MutSß, MutL{alpha} and MutS) and WRN. The MMR proteins were pre-incubated with the DNA substrate on ice for 1 min prior to the addition of WRN. The reactions were incubated at 37°C for 30 min and terminated by the addition of 0.5 reaction volume of buffer S [150 mM EDTA, 2% (w/v) SDS, 30% (v/v) glycerol, 0.1% (w/v) bromophenol blue] followed by treatment with proteinase K (0.1 mg/ml) at 37°C for 10 min. The reaction products were resolved on a non-denaturing 10% polyacrylamide gel [acrylamide to bis-acrylamide, 19:1 (w/w)] run in 1xTBE buffer at 140 V. Radiolabeled DNA species were visualized by a phosphorimager and quantified using ImageQuant software (Molecular Dynamics).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Physical association between WRN and MMR proteins
Based on genetic studies in yeast, it has been suggested that the proteins involved in the initiation of MMR act in conjunction with RecQ DNA helicases to eliminate DNA recombination intermediates containing mismatches, which can give rise to chromosomal rearrangements (27,28). We sought to test the validity of this model biochemically using the WRN helicase, one of the five RecQ homologs identified in human cells, whose dysfunction results in chromosomal translocations and deletions. First, we performed an ELISA-based protein-binding assay to investigate whether WRN and the MMR proteins interact physically. Increasing concentrations of purified MutS{alpha} (MSH2/MSH6), MutSß (MSH2/MSH3) and MutL{alpha} (MLH1/PMS2) proteins (Figure 1A) ranging from 0 to 80 nM were incubated in wells that had been pre-coated with WRN at a concentration of 20 nM and subsequently blocked with BSA to prevent non-specific interactions. After extensive washing, the bound MMR proteins were incubated with specific antibodies followed by a colorimetric assay to quantify the binding. In control experiments, MMR proteins were incubated in wells pre-coated only with BSA. We found that all the three MMR heterodimers were specifically bound to WRN-coated wells in a dose dependent manner, indicating a direct interaction (Figure 1B–D). Interestingly, the apparent dissociation constant of the MutSß–WRN complex (Kd = 8.8 nM) was much lower than that estimated for the MutS{alpha}–WRN complex (Kd = 38.5 nM). The dissociation constant of the MutL{alpha}–WRN complex (Kd = 34.9 nM) was similar to that of the MutS{alpha}–WRN complex.


Figure 1
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  		Figure 1. Direct physical interaction of WRN with MMR proteins. (A) SDS–PAGE analysis of purified recombinant MutS{alpha}, MutSß, MutL{alpha} and WRN proteins produced in insect cells by means of the baculovirus system. (B) Binding of MutS{alpha} to WRN as a function of MutS{alpha} concentration. (C) Binding of MutSß to WRN as a function of MutSß concentration. (D) Binding of MutL{alpha} to WRN as a function of MutL{alpha} concentration. Increasing concentrations of MutS{alpha}, MutSß and MutL{alpha} (0–80 nM) were incubated at 37°C for 1 h in the wells of an ELISA plate that were pre-coated with the WRN protein (20 nM) and subsequently blocked with 3% BSA. After extensive washing, the bound MMR proteins were detected by ELISA as described in Materials and Methods. The measured absorbance values were corrected by subtracting background values obtained with BSA-coated wells. The data points represent the mean of three independent experiments.

 
To test whether WRN and MMR proteins form a stable complex in vivo, we immunoprecipitated WRN from extracts of exponentially growing human embryonic kidney cells (HEK 293) and subjected the resulting immunoprecipitate to western blot analysis. This immunoprecipitate was found to contain the MLH1 and PMS2 proteins, components of the MutL{alpha} complex, but not the MSH2 and MSH6 proteins, which form the MutS{alpha} heterodimer (Figure 2A, lanes 3 and 4). None of these MMR proteins were detected in the immunoprecipitate obtained with control IgGs (Figure 2A, lane 2). To exclude the possibility that the observed association of WRN with MLH1 and PMS2 results from independent binding of these proteins to DNA, we pre-treated the cell extracts with DNaseI. We found that this treatment did not alter the level of the MMR proteins in the WRN immunoprecipitate, suggesting that the WRN–MLH1–PMS2 complex is mediated by protein–protein interactions (Figure 2A, compare lanes 3 and 4). Furthermore, we did not detect PMS2 in an immunoprecipitate obtained with anti-WRN antibody from extracts of the WS cell line AG11395, excluding the possibility that the observed co-immunoprecipitation of MMR proteins with WRN is due to cross-reactivity of the antibody (Figure 2B).


Figure 2
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  		Figure 2. WRN forms a stable complex with MLH1 and PMS2 in human cells. (A) Western blot analysis of WRN immunoprecipitates from total extracts of HEK 293 cells (1.6 mg of protein) before (lane 3) and after (lane 4) treatment with DNaseI (50 U). Lane 1, 5% of input; lane 2, control immunoprecipitation experiment with a preimmune rabbit IgGs. Purified rabbit anti-WRN IgGs (2 µg) were used to immunoprecipitate WRN. Blots were probed with monoclonal antibodies against WRN, MSH2, MSH6, MLH1 and PMS2. (B) Western blot analysis of immunoprecipitate from extracts of AG11395 (WRN–/–) cells obtained using an anti-WRN antibody. The immunoprecipitations were carried out under the same conditions as in (A). The blots were probed with a monoclonal antibody against PMS2.

 
Collectively, these data indicate that MutL{alpha} but not MutS{alpha}, forms a stable complex with WRN in vivo.

Mapping of protein–protein interaction domains
To identify the MutS{alpha}, MutSß and MutL{alpha}-interaction sites on WRN, we performed affinity pull-down assays using a series of WRN fragments fused to GST. These fragments covered the entire WRN polypeptide except for the first 50 amino acids and the region spanning the amino acids 450–499 (Figure 3A and B). The GST pull-down experiments revealed that the MutS{alpha} interaction site on WRN was localized to the region between amino acids 500 and 946, which constitutes the helicase core of WRN composed of the DExH helicase and Zn-binding domains (Figure 3A and C). MutSß was found to make contacts not only with the helicase core of WRN, but also with a region spanning amino acids 947–1149 that contains the winged-helix (WH) motif, a common interaction site for most of the WRN partners identified thus far (Figure 3A and C) (39). Notably, the binding affinity of MutSß to the WH domain of WRN appeared to be much higher that its binding affinity to the helicase core of WRN (Figure 3C, compare lanes 4–7). The data also indicated that MutSß binds to WRN more efficiently than MutS{alpha} (Figure 3C, top and middle panels; compare lane 1 with lanes 4–7), which is in agreement with the results of the ELISA assay (Figure 1).


Figure 3
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  		Figure 3. Mapping of MutS{alpha}, MutSß and MutL{alpha}-interaction domains on WRN (A) Domain organization of the WRN protein. Exo, exonuclease domain; DExH, helicase domain; Zn, zinc-binding domain; WH, winged-helix domain; HRDC, helicase and RNaseD C-terminal domain; RQC, RecQ C-terminal region. The black lines indicate the boundaries of the various WRN fragments used in this study. (B) SDS–PAGE analysis of GST–WRN fragments expressed in E. coli and isolated using glutathione beads. Bands migrating below the WRN fragments (marked by an asterisks) were determined to be degradation products by western blot analysis using an anti-GST antibody. (C) GST pull-down assay. Glutathione beads coated with the indicated GST-tagged WRN fragments were incubated with purified MutS{alpha}, MutSß or MutL{alpha} proteins expressed in insect cells using baculovirus system and the bound MMR proteins were analyzed by western blotting as described in Materials and Methods.

 
MutL{alpha} was found to interact with the helicase core of WRN and with the N-terminal portion of WRN including the exonuclease domain, showing a higher binding affinity to the former domain (Figure 3A and C, bottom panel; compare lanes 3–7).

To identify the subunits of MutS{alpha}, MutSß and MutL{alpha} that mediate the interaction with WRN, we performed a quantitative YTHassay with the full-length WRN as prey. The following interactions were examined: MSH2–WRN, MSH6–WRN, MSH3–WRN and MLH1–WRN. We found WRN to interact with MLH1 and MSH2, but not with MSH3 and MSH6 (Figure 4A and B). This indicates that the MutS{alpha}–WRN and MutSß–WRN interactions are mediated by MSH2, and the MutL{alpha}–WRN interaction is mediated by MLH1. However, the inability of MSH3 and MSH6 to interact with WRN in the YTH assay could be a consequence of the fact that these proteins are not soluble when expressed alone (33). This is also true for PMS2 (34). Therefore, the possibility still exists that these proteins could make additional contacts with WRN. This is particularly likely in the case of MSH3, since our GST pull-down experiments revealed that MutSß interacts with both the helicase core and the WH domain of WRN, whereas MutS{alpha} interacts only with the helicase core of WRN (Figure 3).


Figure 4
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  		Figure 4. Interaction between WRN and MMR proteins in yeast two-hybrid system. (A) Domain organizations of the MSH2 and MLH1 proteins. The numbers refer to the amino acid sequence. (B) Quantitative yeast two-hybrid assay. The S. cerevisiae strain L40 harboring a pACT2 vector expressing the full-length WRN fused to the GAL4 activation domain was transformed with pBTM116 vectors encoding the indicated MMR proteins fused to LexA (MLH1 and its deletion derivatives, MSH3 and MSH6) or Gal4 (MSH2) DNA binding domains. Clones containing both plasmids were subjected to ß-galactosidase assay on an ELISA micro-plate using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) as a substrate. Top panel: An ELISA plate after a 30-min incubation at room temperature. Bottom panel: Graph showing absorbance at 615 nm measured in individual wells after 35 min, which is a measure of ß-gal activity. The values represent the mean of three independent experiments.

 
In order to identify the WRN interaction domain on MLH1, we tested a series of MLH1 deletion variants for the ability to interact with the full-length WRN in YTH assay. We found that this domain is located at the C-terminus of the MLH1 polypeptide between amino acids 500 and 756 (Figure 4A and B). This is different from the location of the BLM-interaction site that was mapped to the region spanning amino acids 396–500 (29).

Stimulation of the helicase activity of WRN by MutS{alpha} and MutSß
Next, we tested MutS{alpha} for the ability to affect the helicase activity of WRN on DNA substrates containing mismatches. In these experiments, we used a synthetic DNA duplex (49 bp) with a 3'-ss flap (19 nt) resembling a part of the structure that results from annealing of the resected arms of a broken chromosome at regions of homology. On such forked DNA structures, WRN preferentially translocates along the 3'-flap oligonucleotide to unwind the duplex ahead of the fork junction, generating a 3'-tailed duplex. This primary product can be further unwound by WRN into the component strands, as a consequence of loading of a second helicase molecule on the 3'-ssDNA tail (40). We prepared a fully matched substrate and a substrate containing a single G/T mismatch located 11 nt ahead of the ss/ds junction (Figure 5A and B, top panels). WRN alone displayed a very low helicase activity on both structures when present at the same concentration as the DNA substrate (1 nM). However, the helicase activity of WRN on these structures dramatically increased upon inclusion of an 8-fold molar excess of MutS{alpha} in the reaction (Figure 5A–D). Notably, the initial rate of the MutS{alpha}-stimulated unwinding reaction with G/T substrate was about 1.7 times higher than that measured with the G/C substrate (Supplementary Table 2).


Figure 5
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  		Figure 5. Kinetics of WRN-mediated unwinding of a 3'-flap DNA duplex in the presence of MutS{alpha} and MutL{alpha}. (A) Reactions with a homoduplex 3' flap substrate. (B) Reactions with a 3'-flap duplex containing a G/T mismatch located 11 nucleotides ahead of the fork as indicated. (C) Quantification of the time course reactions in A. (D) Quantification of the time course reactions in B. All reactions contained 1 nM [32P]DNA, 1 nM WRN and 8 nM MMR proteins as indicated. Aliquots at individual time points were analyzed by native PAGE followed by phosphorimaging and quantification as described in Materials and Methods section. Schemes of the substrate and the reaction products are shown on the right. The relative concentration of unwound products (3'-tailed duplex and free labeled oligonucleotide) is expressed as a percentage of total DNA.

 
Since MutL{alpha} is known to bind to MutS{alpha}–heteroduplex complexes (41), we investigated whether it can affect the WRN-mediated unwinding of G/T and G/C substrates induced by MutS{alpha}. We found that MutL{alpha} did not significantly alter the MutS{alpha}-dependent helicase activity of WRN on these DNA structures (Figure 5). Also, it had no effect on WRN-mediated unwinding in the absence of MutS{alpha} (Figure 7, lane 5).

To further assess the effect of MutS{alpha} on WRN-mediated unwinding of the 3'-flap duplex, we performed a protein titration experiment, in which we varied the concentration of MutS{alpha} while keeping WRN and DNA substrate at a fixed concentration of 1 nM. We found that MutS{alpha} stimulated the helicase activity of WRN in a concentration-dependent manner, exhibiting a significantly higher activity on the G/T substrate than on the homoduplex substrate (Figure 6).


Figure 6
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  		Figure 6. Helicase activity of WRN on 3'-flap duplexes with or without a G/T mismatch as a function of MutS{alpha} concentration. WRN and the DNA substrate were present at a concentration of 1 nM. The DNA substrates were the same as in Figure 5. Reactions were incubated for 30 min and analyzed by native PAGE followed by phosphorimaging as described in Materials and Methods section. The data points represent the mean of three independent experiments. The relative concentration of unwound products (3'-tailed duplex and free labeled oligonucleotide) is expressed as a percentage of total DNA.

 
To explore the specificity of the observed stimulatory effect, we tested human MutSß as well as E. coli MutS for the ability to stimulate DNA unwinding by WRN. We found that MutSß enhanced the WRN-mediated unwinding of the 3'-flap DNA duplex to a similar extent as seen with MutS{alpha} (Figure 7, compare lanes 2 and 3). In contrast, the E. coli MutS protein did not enhance the WRN-mediated DNA unwinding (Figure 7, lane 8), indicating that the observed stimulatory effect is specific to human MutS homologs. As in the case of MutS{alpha}, MutSß-stimulated helicase activity of WRN was not influenced upon addition of MutL{alpha} (Figure 7, compare lanes 4 and 7) and it was dependent on MutSß concentration (Supplementary Figure S1). We also found that in the presence of MutSß, WRN unwound the G/T substrate with the same efficiency as the homoduplex substrate (Supplementary Figure S2). This is consistent with the fact that MutSß does not bind to base–base mismatches (22) and supports the conclusion that the observed stimulatory effect of the G/T mismatch on MutS{alpha}-dependent unwinding of the 3'-flap duplex by WRN results from the specific binding of MutS{alpha} to the mismatch.


Figure 7
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  		Figure 7. Effect of various MMR proteins on WRN-mediated unwinding of a 3'-flap DNA duplex. Helicase reactions contained 1 nM [32P]DNA substrate and 2 nM WRN. The MMR proteins were present in a concentration of 8 nM as indicated. Reactions were incubated for 32 min and analyzed by native PAGE followed by phosphorimaging as described in Materials and Methods section (top panel). The relative concentration of unwound products (3'-tailed duplex and free labeled oligonucleotide) is expressed as a percentage of total DNA (bottom panel). Schemes of the substrate and the reaction products are shown on the right.

 
Together, the results described above indicate that MutS{alpha} and MutSß, but not MutL{alpha}, can stimulate WRN to unwind DNA structures resembling recombination intermediates and that this stimulatory effect is enhanced by mismatches in the DNA substrate.

MutS{alpha} and MutSß stimulate WRN helicase specifically on forked DNA duplexes with a 3'-ss arm
To gain further insights into the mechanism underlying the stimulation of the helicase activity of WRN by MutS{alpha} and MutSß, we investigated the dependence of this reaction on the configuration of the arms of the fork. Using the same set of oligonucleotides, we prepared the following substrates: a forked duplex with both arms single stranded (splayed arm); a forked duplex with the 3'-arm single stranded and the 5'-arm double stranded (3'-flap duplex); a forked duplex with the 3'-arm double stranded and the 5'-arm single stranded (5'-flap duplex) and a forked duplex with both arms double stranded. Earlier studies revealed that WRN could unwind efficiently all these structures, indicating that it does not require the 3'-arm to be single stranded for loading at the fork (40). We found that MutS{alpha} and MutSß strongly stimulated the WRN-mediated unwinding of the splayed arm and the 3'-flap duplex, but had no significant effect on the unwinding of the 5'-flap duplex and the fully double stranded fork (Figure 8). We also tested these proteins for the ability to stimulate the helicase activity of WRN on 3'-ssDNA-tailed duplex, which is normally a poor substrate for WRN (40). We found that neither MutS{alpha} nor MutSß could activate WRN for unwinding of this partial DNA duplex (data not shown). Interestingly, the 3'-tail duplex resulting from unwinding of the 3'-flap structure was unwound by WRN efficiently. This discrepancy can result from the fact that WRN exists as an oligomeric structure, which would facilitate loading of a second molecule of WRN on the 3'-ssDNA generated by unwinding of the duplex ahead of the fork.


Figure 8
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  		Figure 8. Stimulation of WRN-mediated unwinding of forked DNA structures by MutS{alpha} and MutSß is dependent on the presence of 3'-single stranded DNA at the junction. Reactions with 1 nM splayed arm (white bars), 1 nM 3'-flap duplex (light gray bars), 1 nM 5'-flap duplex (dark gray bars) and 1 nM fully double-stranded fork duplex (black bars) contained 1 nM or 4 nM WRN and 8 nM MutS{alpha} or MutSß as indicated. Reactions were incubated for 32 min and analyzed by native PAGE followed by phosphorimaging as described in Materials and Methods. The relative concentration of unwound products is expressed as a percentage of total DNA. The data points represent the mean of at least three independent experiments.

 
Collectively, these data indicate that MutS{alpha} and MutSß require a forked DNA structure with 3'-ss DNA at the junction to stimulate WRN-mediated DNA unwinding.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY DATA
 REFERENCES
 
Although WRN has been implicated in a number of DNA repair processes, the exact DNA transactions mediated by this helicase/exonuclease in the cell remain elusive. Here we show that WRN interacts physically with proteins that are involved in the initiation of MMR and the rejection of recombination between divergent sequences. Most importantly, our experiments revealed that MutS{alpha} and MutSß can stimulate the helicase activity of WRN on forked DNA structures with a 3'-ss arm that resemble intermediates of single-strand annealing pathways of HR. In addition, we found that a single G/T mismatch located ahead of the fork junction increased the efficiency of the MutS{alpha}-dependent unwinding by WRN. These data are consistent with a model in which the MMR initiation factors prevent homeologous recombination by activating a DNA helicase for unwinding of recombination intermediates containing mismatches. This model was proposed earlier on the basis of results of in vivo heteroduplex rejection assays with yeast msh2 and sgs1 mutants (27,28). It is possible that MutS{alpha} and MutSß bind to mismatches formed after pairing of sequences of imperfect homology and, following ATP binding, are converted into a DNA sliding clamp as proposed in the case of the MMR pathway (42). When the clamp encounters the junction between the heteroduplex and the non-homologous 3'-SS tail, it binds stably to it and recruits a DNA helicase to disrupt the joined DNA molecule. In agreement with this hypothesis, it has been demonstrated that yeast MutSß specifically binds to forked DNA structures containing 3'- ssDNA making contacts with the sequences at the ds–ss junction (43). These studies also revealed that MutSß holds the junction in an altered, perhaps more rigid, conformation (43). Such structural changes could facilitate the loading of the WRN helicase on the 3'- ssDNA at the junction, which is a prerequisite for duplex unwinding to occur. However, it should be noted that the MutS{alpha}-activated unwinding of a 3'-flap duplex by WRN displayed only a moderate dependence on mismatches. It is therefore possible that hereroduplex rejection in vivo involves some additional factors that ensure mismatch specificity of this transaction.

In our studies, we did not observe any significant modulation of WRN-mediated unwinding by MutL{alpha}, even in the presence of MutS{alpha} or MutSß. In agreement with this finding, the yeast Mlh1 and Pms1 proteins have been shown to have only minor roles in the rejection of homeologous recombination relative to the contributions of Msh2 and Msh6 (27). Thus, it appears that the physical interaction between WRN and MutL{alpha} identified in this study has some other functional implication. Interestingly, MLH1 was shown to interact with various DNA repair factors including MRE11, BACH1, MBD4 and BLM (29,44–47). It is, therefore, possible that MLH1 plays a more general role in DNA repair processes.

A number of other functional implications for the observed interactions between WRN and the MMR factors can be discussed. Several lines of evidence suggest that WRN promotes replication of telomeric DNA by unwinding G-quadruplex structures that can readily form in G-rich telomeric DNA and impose a barrier for progression of DNA replication forks (9,48–50). Strikingly, human MutS{alpha} has been shown to bind efficiently to G-quadruplex DNA (51). Moreover, Msh2 deficiency in mice is associated with loss of telomeres and an elevated level of telomere end-to-end fusion, a phenotype similar to that manifested by WRN-deficient cells (9,52). Thus, one can speculate that the MMR proteins can mediate recruitment of the WRN helicase to G-quadruplex structures formed at telomeres and hence facilitate their removal.

It has been shown that the human MutSß and WRN are required along with PCNA, RPA and ERCC1-XPF for uncoupling of psoralen-induced inter-strand DNA crosslinks (ICLs) in cell-free extracts, suggesting a novel ICL-repair pathway in which MutSß is essential for the recognition of ICLs, while the WRN helicase mediates unwinding of the DNA duplex adjacent to the lesion, which enables strand incision by ERCC1-XPF (53,54). Our finding that MutSß physically interacts with WRN and stimulates its helicase activity brings further support for this model and suggests that MutSß might recruit WRN to the ICL sites.

Earlier studies demonstrated that nuclear extracts from several fibroblastoid cell lines derived from WS patients were deficient in repair of base–base mismatches and insertion/deletion loops, suggesting that WRN could have a role in MMR (55). However, it is not certain that the MMR-deficiency of these extracts was caused solely by WRN deficiency because complementation experiments with recombinant WRN protein were not performed in this study. Moreover, in some cases, pair-wise mixing of these extracts restored MMR proficiency, making the involvement of WRN in MMR rather questionable.

Recently, two other human RecQ homologs, namely RECQ1 and BLM, have been shown to interact physically and functionally with the MMR-initiation factors (29,31,47,56,57). As in the case of WRN, MutS{alpha} was found to stimulate RECQ1-mediated unwinding of a forked structure with a 3'-ss arm (56). In contrast, MutS{alpha} did not affect unwinding of forked DNA duplexes by BLM (31). Instead, MutS{alpha} was found to stimulate the ability of BLM to process Holliday junctions in vitro (57). Further studies will be needed to fully understand the molecular mechanisms by which the RecQ helicases and MMR factors work together to maintain genomic stability.


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
REFERENCES
 
Supplementary Data are available at NAR Online.


   ACKNOWLEDGEMENTS
 
We thank Renjie Jiao for the construction of pACT2-WRN plasmid used in YTH assay, Peter Cejka for purified E. coli MutS protein and Lene Rasmussen for the plasmid pLJR105. We are also grateful to Josef Jiricny, Stefano Ferrari and David Lauterbach for comments on the manuscript. This work was supported by the Sassella and the Swiss National Science Foundations (Marie Heim-Vögtlin Grant Nr. PMPDA-102451 to N.S.). Funding to pay the Open Access publication charges for this article was provided by the Swiss National Science Foundation.

Conflict of interest statement. None declared.


   Footnotes
 
Present address: Nurten Saydam, Harvard School of Public Health, 667, Huntington Ave, Boston, USA.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.


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 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 

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