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Human Molecular Genetics Advance Access originally published online on July 28, 2004
Human Molecular Genetics 2004 13(19):2247-2261; doi:10.1093/hmg/ddh234
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Human Molecular Genetics, Vol. 13, No. 19 © Oxford University Press 2004; all rights reserved

In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication

Sudha Sharma1,2, Joshua A. Sommers1 and Robert M. Brosh, Jr1,*

1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA and 2Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

Received May 7, 2004; Accepted July 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase–endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN–FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We have been interested in understanding how the WRN gene product, which is defective in the premature aging disorder Werner syndrome (WS) (1), functions at the molecular and cellular level in pathways that are important for the maintenance of genomic stability. The replication defects (2–4) and hypersensitivity of WS cells to DNA damaging agents (5–8) might suggest a direct role of WRN protein, a helicase (9,10) and exonuclease (11), in the processes of replication and/or repair. Although a number of WRN protein interactors have been identified and characterized (12,13), the biological importance of any of these interactions remains to be demonstrated. We have been particularly interested in the protein interaction between WRN and flap endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA replication and repair (14). The robust stimulation of FEN-1 cleavage by WRN (15,16) raises the possibility that the two proteins function together in cellular pathway(s) to maintain genomic stability.

To address the biological significance of the WRN–FEN-1 interaction, we selected Saccharomyces cerevisiae as a model genetic system because human FEN-1 (hFEN-1) and yeast FEN-1 (yFEN-1) encoded by rad27 have conserved functions (17). The cooperative and overlapping roles of yFEN-1 and Dna2 guided us to examine the ability of WRN to rescue the replication defects of a dna2 mutant background (18). A compensatory relationship between yFEN-1 and Dna2 is likely to exist because a rad27 deletion with a dna2 mutation is synthetically lethal and overexpression of either nuclease under the control of a galactose-inducible promotor from a multi-copy plasmid complements the mutant phenotypes associated with the single mutant (19). Emerging evidence indicates that Dna2, a helicase (20) and endonuclease (18,21), participates with FEN-1 in Okazaki fragment processing. yFEN-1 and Dna2 have been shown to co-purify and physically interact with each other (19). Recently it was proposed that Dna2 and FEN-1 sequentially act together during DNA replication on 5' flap replication intermediates in a manner that is governed by the single-stranded DNA binding protein RPA (22). Dna2 cleaves long (~30 nt) 5' RNA–DNA chimeric flaps beyond the terminal RNA segment within the DNA flap. A model was proposed in which the 5' flap generated by polymerase strand displacement becomes sufficiently long for RPA to bind (22). RPA bound to the 5' ssDNA flap inhibits FEN-1 cleavage of the flap structure, but allows Dna2 to endonucleolytically cleave. The shorter 5' flap product, which no longer efficiently binds RPA, can then be subsequently acted upon by FEN-1 or a related nuclease such as exonuclease 1 (EXO-1).

Genetic and biochemical evidences suggest that the function of Dna2 in vivo might be to compensate for FEN-1 cleavage under certain circumstances when the FEN-1 cleavage reaction is inhibited (23,24), perhaps by DNA structure. The 5' to 3' helicase activity of Dna2 removes secondary structure within the 5' flap enabling FEN-1 to load on the free 5' end of the flap and cleave the structure catalytically at the base of the flap (25). In vitro studies indicate that the size of the displaced strand may be modulated by the concerted action of polymerase {delta}, PCNA, RPA and FEN-1 (26). The curtailed ability of FEN-1 to cleave long 5' flap substrates (27) is thought to reflect the mechanism of FEN-1 loading and tracking from the 5' ssDNA end to the base of the 5' flap structure in an energy-independent reaction (28,29).

Fluorescence resonance energy transfer (FRET) analyses show that WRN and FEN-1 form a complex in vivo that co-localizes in foci associated with arrested replication forks (30). Biochemical studies demonstrate that WRN effectively stimulates FEN-1 cleavage of branch-migrating double-flap structures that are the proposed physiological substrates of FEN-1 during replication (30). Kinetic analyses of the FEN-1 cleavage reaction suggest that WRN improves the efficiency of the FEN-1 cleavage reaction by a mechanism distinct from that of PCNA (15). Stimulation of FEN-1 cleavage by WRN is mediated by a protein interaction and does not require WRN catalytic activity (16). We reasoned that WRN might stimulate yFEN-1 cleavage in vivo to rescue the DNA replication and repair phenotypes of the dna2 mutant. The results presented here support this hypothesis and provide evidence for the biological importance of the WRN–FEN-1 interaction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
WRN rescues the temperature-sensitive growth phenotype of a yeast dna2 replication mutant
A yeast dna2-1 mutant strain transformed with a multi-copy URA3 selectable plasmid encoding either full-length human WRN protein (residues 1–1432) or a WRN non-catalytic C-terminal fragment (residues 940–1432) was tested for inducible protein expression in the presence of 2% galactose (gal) by western blot analysis. Both full-length WRN and WRN940–1432 (Fig. 1A) were detected after 1.5 h gal induction and greater amounts were detected after 3 or 4.5 h induction. Quantitative western blot analyses using purified recombinant WRN or WRN940–1432 protein as standard indicated that after 4.5 h induction by 2% gal, ~4.3x106 or 5.2x106  molecules/cell of WRN or WRN940–1432, respectively, were present (data not shown). In comparison, we determined that the level of endogenous WRN in HeLa cells was 8.9x104 molecules/cell (data not shown), in agreement with previously published values for WRN copy number in other human cells (31,32). A dna2-1 strain that had been transformed with empty vector (dna2-1/vector) did not express protein specifically recognized by WRN antibody (Fig. 1A).



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  		Figure 1. Genetic complementation of ts growth of dna2-1 by WRN overexpression. (A) Expression of human WRN in a yeast dna2-1 mutant strain. Yeast dna2-1 mutant cells transformed with YEp195SpGAL, YEp195SpGAL–WRN, or YEp195SpGAL–WRN940–1432 were grown in SC gal minus Ura media. Cells were harvested at 0, 1.5, 3 or 4.5 h after gal induction and extracts were prepared by alkaline lysis. Equal amounts of total cell lysate were loaded on 10% polyacrylamide SDS gels followed by western blot detection using anti-WRN antibody. Purified recombinant full-length WRN (His–WRN) and WRN940–1432 (His–WRN940–1432) were included as controls. (B) Transformed yeast dna2-1 strains were streaked onto SC gal minus Ura plates along with the parental wild-type DNA2/vector strain to serve as a positive control. Plates were incubated at 23 or 37°C.

 
A dna2-1 mutant strain is characterized by a temperature-sensitive (ts) growth defect at the non-permissive temperature of 37°C (18). To assess the effect of WRN expression on the ts phenotype of the dna2-1 strain, transformed dna2-1 cells were streaked onto synthetic complete (SC) gal minus uracil (Ura) plates and allowed to grow at either permissive (23°C) or restrictive (37°C) temperature. Because overproduction of RAD27 is reported to suppress the ts growth of dna2-1 mutants (19), we have included this as positive control in our experiments. The dna2-1 strain expressing either WRN or WRN940–1432 grew at 37°C similar to dna2-1 expressing RAD27 and comparable to the wild-type DNA2/vector, whereas dna2-1/vector grew poorly at 37°C (Fig. 1B). All the transformed strains grew normally at 23°C. None of the strains grew on plates containing the drug 5-fluoroorotic acid (5-FOA) at 37°C (data not shown), confirming that complementation of the ts phenotype is indeed conferred by the expression plasmid rather than by some rearrangement/mutations in the dna2-1 cells. The ability of WRN940–1432 to rescue the ts growth defect of dna2-1 indicates that WRN ATPase/helicase or exonuclease activities are not required for complementation of the mutant replication phenotype.

WRN expression rescues the ts cell cycle arrest of a dna2-1 mutant
The ability of WRN to restore viability to dna2-1 mutant cells at 37°C prompted us to examine the effect of WRN expression on cell cycle progression at the restrictive temperature. The dna2-1 mutant cells are reported to arrest at 37°C with 2C DNA content and display a large proportion of large-budded cells (18,33). Morphological examination of dna2-1/vector cells that had been incubated for 4 h at 37°C indicated that ~75% of the cells were arrested as large-budded cells (‘dumbbell’) with an undivided nucleus positioned at or near the bud neck (Fig. 2A). In contrast, 27% of transformed wild-type DNA2/vector cells were dumbbell shaped. The percentage of dumbbell shaped dna2-1 cells expressing WRN or WRN940–1432 was determined to be 32 and 33%, respectively, a level comparable to the parental DNA2 strain (Fig. 2A). These results indicate that expression of either full-length WRN or WRN940–1432 in dna2-1 mutant cells restored the near normal cell division cycle when incubated at 37°C.



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  		Figure 2. WRN rescues the G2/M arrest in dna2-1 mutant cells. Transformed dna2-1 strains were grown at 23°C in SC raffinose minus Ura to a density of 1.5x106 cells/ml. Cultures were then spun down, resuspended in SC gal, split and incubated at either 23 or 37°C for 4 h. Cells were subsequently processed for DAPI staining and FACS analysis. The parent DNA2/vector strain was included as a positive control. (A) Composite images of DIC and fluorescence (DAPI staining) of the transformed yeast strains that had been incubated at permissive or restrictive temperature is shown. Percent of cells with the large-budded (dumbbell) phenotype is also shown. (B) Transformed yeast cells were processed for SyTOX green staining and flow cytometric analysis as described in Materials and Methods. DNA content of the various yeast strains incubated at 23 or 37°C is shown.

 
After incubation for 4 h at 37°C, a greater fraction of dna2-1/vector cells had 2C DNA content when compared with dna2-1/vector cells incubated at 23°C (Fig. 2B). These results, as previously reported (33), indicate that the dna2-1 cells were arrested at the G2/M border with replicated DNA and suggest an accumulation of S-phase errors. Expression of WRN resulted in a comparable DNA content profile at 23 and 37°C, as observed for the DNA2/vector strain (Fig. 2B). However, the rescue of the dna2-1 cell cycle arrest was not comparable for cells expressing WRN versus WRN940–1432. Although expression of WRN940–1432 rescued the G2 arrest/delay, there was still a reproducible delay in S phase. These results suggest that WRN catalytic activity may be required for proper S phase progression but not essential for preventing the G2 delay.

WRN complements the sensitivity of dna2-1 to hydroxyurea or methylmethane sulfonate
In addition to the ts replication defect, dna2-1 mutant cells are sensitive to hydroxyurea (HU) (34), an agent that blocks replication by depleting the nucleotide pool. To assess the ability of WRN to rescue the HU sensitivity of the dna2-1 mutant, 10-fold serial dilutions of exponentially growing strains were spotted onto SC gal minus Ura plates with or without 150 mM HU. As expected, dna2-1/vector cells were highly sensitive to HU (Fig. 3A). Mutant dna2-1 cells expressing either WRN or WRN940–1432 were resistant to the negative effect of HU on cell viability (Fig. 3A). We conclude that WRN effectively rescued HU sensitivity of the dna2-1 mutant, and this complementation did not require WRN catalytic activities.



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  		Figure 3. WRN complements the HU or MMS sensitivity of a dna2-1 mutant strain. Ten-fold serial dilutions of exponentially growing yeast cultures of dna2-1 strains were plated on SC minus Ura plates containing 2% gal with or without 150 mM HU (A) or 0.006% MMS (B) and grown for 5–7 days at 23°C. Control plates were incubated for 3–5 days. Wild-type DNA2/vector strain was included as a positive control. (C) Expression of WRN/WRN940–1432 can be regulated by gal concentration. WRN/WRN940–1432 expression in the transformed dna2-1 cells was induced with the indicated gal concentrations and the cells were harvested 8 h after induction. Equal amounts of total cell lysate were loaded on 10% polyacrylamide SDS gels followed by western blot detection using anti-WRN antibody. Purified recombinant full-length WRN (His–WRN) and WRN940–1432 (His–WRN940–1432) proteins were included as controls. (D) Reduced levels of WRN or WRN940–1432 partially rescue the MMS sensitivity of the transformed dna2-1 mutant cells. Five-fold serial dilutions of exponentially growing cultures of the transformed dna2-1 strains were spotted onto SC minus Ura plates containing the indicated gal concentrations in the presence or absence of 0.006% MMS.

 
The dna2-1/vector strain was sensitive to a low dose (0.006%) of the DNA alkylating agent methylmethane sulfonate (MMS) compared to DNA2/vector (Fig. 3B), as previously reported (34,35). The induced expression of either WRN or WRN940–1432 in the dna2-1 background by 2% gal suppressed the MMS sensitivity compared to dna2-1/vector cells (Fig. 3B). Previous studies have shown that gene expression levels from the GAL1/10 promotor can be regulated by altering the concentration of gal in the growth medium (36). We therefore examined the level of WRN/WRN940–1432 protein expression in cells grown at lower gal concentrations (0–2%) in the presence of 2% raffinose. The western blot results, shown in Figure 3C, demonstrated that indeed WRN/WRN940–1432 expression was dependent on the concentration of gal in the medium. With respect to WRN protein level at 2% gal, 89, 69 and 31% WRN were detected at 0.5, 0.05 and 0.005% gal, respectively, in the medium. Similarly, 85, 57 and 23% WRN940–1432 were detected at 0.5, 0.05 and 0.005% gal, respectively, with respect to the level of WRN940–1432 at 2% gal. In the media containing 2% raffinose but lacking gal, a very low level of WRN or WRN940–1432 was consistently detected when extracts from a greater number of yeast cells were used for western detection (data not shown). As WRN expression could be regulated by the level of gal, we wanted to determine if genetic complementation of dna2-1 might be achieved at lower levels of WRN/WRN940–1432 expression. The MMS sensitivity was partially rescued by either WRN or WRN940–1432 without gal induction when the cells were grown on 2% raffinose (Fig. 3D). Increased levels of gal resulted in greater complementation of the MMS sensitivity for the WRN or WRN940–1432 transformed strains. Partial complementation of the HU and ts phenotypes of the dna2-1 mutant by WRN or WRN940–1432 was also observed at lower gal levels (data not shown).

WRN stimulates yeast FEN-1 cleavage of its preferred double-flap substrate
Overproduction of yFEN-1 suppressed the mutant cellular phenotypes of the dna2-1 strain (19). The ability of WRN to stimulate hFEN-1 cleavage activity on DNA flap substrates (15,16) suggested that the enhancement of endogenous yFEN-1 cleavage by WRN may be the basis for genetic complementation of the dna2-1 mutant. To explore this hypothesis further, we set out to determine if WRN stimulates yFEN-1 cleavage activity on proposed cellular DNA substrates of FEN-1. yFEN-1 acts during replication upon a double-flap structure with equilibrating 3' and 5' ssDNA tails that arise after strand displacement DNA synthesis by a DNA polymerase (37,38). Initially we tested double-flap substrates with a 1 nt 3' tail because the cleavage specificity of FEN-1 suggests that this is the preferred cellular DNA substrate. yFEN-1 precisely cleaves the double-flap substrate with a 1 nt 3' tail at a position 1 nt into the downstream annealed region to yield the 7 nt product. This cleavage pattern allows the 1 nt 3' tail to anneal and generate a nick suitable for ligation. WRN (100 fmol) resulted in a substantial stimulation of the yFEN-1 cleavage to yield the 7 nt product (Fig. 4A, lanes 2–9 versus lanes 10–17), whereas WRN alone did not cleave this substrate (Fig. 4A, lane 18). At a limiting amount of yFEN-1 (0.625 fmol), only 1.5% of the double-flap substrate was incised (Fig. 4A, lane 3, Fig. 4C). In the presence of WRN, yFEN-1 incision was increased to 32% (~22-fold). Stimulation by WRN was also observed at higher levels of yFEN-1 (Fig. 4C).



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  		Figure 4. WRN stimulates yeast yFEN-1 cleavage of physiological double-flap substrates. Reaction mixtures (20 µl) containing 10 fmol of a double-flap substrate with a 1 nt 3' tail (A) or an equilibrating 3' tail (B), WRN (100 fmol) and/or the specified amounts of yFEN-1 were incubated at 37°C for 15 min under standard conditions. Products were resolved on 20% polyacrylamide 7 M urea denaturing gels. (C) Percent incision for reactions containing the 1 nt 3' tail double-flap substrate and yFEN-1 (open circles) or yFEN-1+WRN (filled circles) or reactions containing the equilibrating 3' tail double-flap substrate and yFEN-1 (open squares) or yFEN-1+WRN (filled squares). (D) A non-catalytic C-terminal domain of WRN retains the ability to stimulate yFEN-1 cleavage of double-flap substrates. yFEN-1 cleavage reactions, as described in (A) and (B), were performed in the presence of WRN940–1432 (100 fmol) and/or the specified amounts of yFEN-1. Products were resolved on 20% polyacrylamide, 7 M urea denaturing gels (Supplementary Material, Fig. 1). the percent incision for reactions containing the 1 nt 3' tail double-flap substrate and yFEN-1 (open circles) or yFEN-1+WRN940–1432 (filled circles) or reactions containing the equilibrating 3' tail double-flap substrate and yFEN-1 (open squares) or yFEN-1+WRN940–1432 (filled squares) is shown.

 
Maturation of Okazaki fragments requires that 5' flaps arising from strand displacement DNA synthesis be processed. As the 5' flaps created are complementary to the template, the DNA intermediate can potentially equilibrate to form configurations of double-flap structures with varying 3' tail and 5' flap lengths. We were interested in how WRN might affect yFEN-1 cleavage on this type of flap structure with overlapping complementary tails capable of equilibration. yFEN-1 cleaved the equilibrating flap substrate to generate the 12 nt cleavage product, indicative of the ability of the substrate to equilibrate to a double-flap structure with a 3' 1 nt tail that can be cleaved by yFEN-1 (37). WRN (100 fmol) stimulated yFEN-1 cleavage of the equilibrating flap substrate over a range of FEN-1 amounts (0.625–20 fmol) (Fig. 4B, lanes 2–9 versus 10–17, Fig. 4C). The greatest stimulation was observed using 1.25 fmol yFEN-1 in which an 11-fold stimulation by WRN was observed. WRN alone did not cleave the substrate (Fig. 4B, lane 18). Consistent with the results presented here, we previously reported that full-length WRN effectively stimulated hFEN-1 cleavage of branch-migrating double-flap structures (30).

As the expression of the WRN940–1432 fragment complemented the dna2-1 mutant phenotypes, we tested the purified recombinant C-terminal WRN fragment for stimulation of yFEN-1. In the absence of WRN940–1432, yFEN-1 (0.625 fmol) cleaved about 1.5% of the double-flap substrate with a 1 nt 3' complementary tail, whereas the yFEN-1 cleavage was stimulated ~32-fold by WRN940–1432 (100 fmol) resulting in 48% incision of the substrate (Fig. 4D; Supplementary Material, Fig. 1A). Stimulation by WRN940–1432 was observed throughout the yFEN-1 titration (Fig. 4D). These results are consistent with our previous finding that the non-catalytic C-terminal WRN domain stimulated hFEN-1 incision of fixed 5' flap and nicked duplex DNA substrates (15,16).

WRN940–1432 also stimulated yFEN-1 incision of the equilibrating double-flap DNA substrate over a range of yFEN-1 amounts (0.625–20 fmol) (Fig. 4D; Supplementary Material, Fig. 1B). The yFEN-1 at 1.25 fmol incised ~2% of the DNA substrate resulting in the 12 nt product. In the presence of WRN940–1432 (100 fmol), 41% of the substrate was cleaved resulting in 20-fold increase in yFEN-1 incision (Fig. 4D; Supplementary Material, Fig. 1B). These results indicate that WRN stimulates yFEN-1 incision of physiological double-flap DNA structures that are associated with DNA replication/repair, and the stimulation does not require the catalytic activities of WRN. WRN940–1432 also effectively stimulated hFEN-1 cleavage of the double-flap substrate with the 1 nt 3' tail or the equilibrating double-flap substrate (data not shown).

Bloom syndrome helicase stimulates FEN-1 cleavage of branch-migrating DNA substrates
During the course of this study, it was reported that ectopically expressed human BLM suppressed the ts growth defect and DNA damage sensitivity of the dna2-1 strain (34). We have reported that BLM stimulates hFEN-1 cleavage of fixed 5' flap and nicked duplex DNA substrates (39). To provide a plausible molecular explanation for the reported genetic complementation of the dna2 phenotypes by human BLM, we investigated a putative interaction between BLM and yFEN-1. BLM stimulated yFEN-1 cleavage of the double-flap substrate with a complementary 1 nt 3' tail to yield the predicted 7 nt product quite significantly throughout the yFEN-1 protein titration (Supplementary Material, Fig. 2A). Using 2.5 fmol of yFEN-1, BLM (100 fmol) stimulated yFEN-1 cleavage by ~12-fold (Fig. 5A). In control reactions, there was minimal substrate cleavage by BLM alone.



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  		Figure 5. BLM stimulates yeast yFEN-1 cleavage of double-flap substrates by a protein interaction. Reaction mixtures (20 µl) containing 10 fmol of a double-flap substrate with a 1 nt 3' tail (A) or an equilibrating 3' tail (B), yFEN-1 (2.5 fmol) and BLM (100 fmol), BLM966–1417 (100 fmol) or MBP (100 fmol) were incubated at 37°C for 15 min under standard conditions. Products were resolved on 20% polyacrylamide, 7 M urea denaturing gels (Supplementary Material, Figs 2 and 3). Percent incision for each indicated set of reactions is shown.

 
A sequence alignment of RecQ family members with the WRN949–1042 protein fragment that mediates the FEN-1 interaction indicated a number of highly conserved residues (16). The region of BLM conserved with the minimal hFEN-1 interaction domain of WRN corresponds to residues 1076–1217 of the BLM protein sequence. We therefore set out to test if a purified recombinant maltose binding protein (MBP)–BLM fusion protein spanning residues 966–1417 outside the core helicase domain of BLM, designated BLM966–1417, might mediate a functional interaction with yFEN-1. BLM966–1417 stimulated yFEN-1 cleavage of the double-flap substrate with a complementary 1 nt 3' tail to yield the predicted 7 nt product in a dose-dependent fashion (Supplementary Material, Fig. 3A). At 2.5 fmol of yFEN-1, incision of the double-flap substrate with a 1 nt 3' tail was increased ~11-fold by 100 fmol of BLM966–1417 (Fig. 5A), a level of stimulation similar to that observed with the full-length BLM protein. The stimulation of yFEN-1 cleavage by BLM966–1417 was specific because MBP did not stimulate yFEN-1 incision.

We next tested the effect of BLM or BLM966–1417 on yFEN-1 cleavage of the equilibrating double-flap substrate. BLM stimulated yFEN-1 cleavage of the equilibrating double-flap substrate to yield the predicted 12 nt product quite significantly throughout the yFEN-1 protein titration (Supplementary Material, Fig. 2B). Using 2.5 fmol of yFEN-1, BLM (100 fmol) stimulated yFEN-1 cleavage by ~12-fold (Fig. 5B). BLM966–1417 also stimulated yFEN-1 incision of the equilibrating double-flap DNA substrate to yield the predicted 12 nt product (Supplementary Material, Fig. 3B). Using 2.5 fmol of yFEN-1, BLM966–1417 (100 fmol) stimulated yFEN-1 cleavage by ~13-fold (Fig. 5B). These results indicate that BLM effectively stimulates yFEN-1 activity by a protein interaction with a region of BLM outside the catalytic ATPase/helicase domain. Furthermore, these results clearly demonstrate that BLM is capable of stimulating yFEN-1 cleavage of its proposed physiological DNA substrates. Full-length BLM or BLM966–1417 (100 fmol) stimulated hFEN-1 cleavage of both double-flap substrates (Fig. 6A and B), indicating that the interaction was similar to that observed for the yFEN-1–BLM pair.



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  		Figure 6. Human FEN-1 cleavage of double-flap substrates is stimulated by BLM protein interaction. Reaction mixtures (20 µl) containing 10 fmol of the double-flap DNA substrate with a 1 nt 3' tail (A) or an equilibrating 3' tail (B), hFEN-1 (0.6 fmol) and BLM (100 fmol), BLM966–1417 (100 fmol) or MBP (100 fmol) were incubated at 37°C for 15 min under standard conditions. Products were resolved on 20% polyacrylamide, 7 M urea denaturing gels. Phosphorimages of typical gels for each substrate are shown.

 
Human WRN or WRN940–1432 expressed in dna2-1 cells interacts with yeast FEN-1
In order to provide evidence to support our hypothesis that the human WRN or WRN940–1432 fragment expressed in dna2-1 cells interacts with yFEN-1, we tested for a physical interaction by applying the appropriate yeast cell extract from transformed dna2-1 cells to immobilized His6-tagged yFEN-1 bound to Ni2+ metal chelate resin. After extensive washing of the yFEN-1 affinity beads, the eluted yeast cell extract proteins were analyzed by western blot. WRN from the dna2-1/WRN cell extract bound to the immobilized yFEN-1 (Fig. 7A, lane 4), but not to the His6 control resin (Fig. 7A, lane 5). Similarly, yeast expressed WRN940–1432 bound specifically to the yFEN-1 beads (Fig. 7A, lane 8), but not to the His6 control resin (Fig. 7A, lane 7). As expected, no signal for WRN or WRN940–1432 was obtained when the dna2-1/vector cell extracts were incubated with either yFEN-1 or His6 beads (Fig. 7A, lanes 3 and 6), attesting to the specificity of WRN detection. Expressions of WRN and WRN940–1432 in dna2-1 are shown (Fig. 7A, lanes 1 and 2). His6–yFEN-1 (325 ng) bound to metal chelate resin used as input was detected by quantitative western blot analysis using an anti-His6 antibody and purified His6–yFEN-1 as standard (Fig. 7A, lanes 3, 4 and 8; data not shown). Control membranes were stained with amido black reagent to detect His6–yFEN-1 and His6 (data not shown). These results demonstrate that yFEN-1 specifically interacts with full-length WRN or the C-terminal WRN940–1432 fragment that was expressed in dna2-1 cells.



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  		Figure 7. WRN or BLM helicases bind to yeast yFEN-1 by their non-catalytic C-terminal domains. (A) WRN or WRN940–1432 expressed in dna2-1 cells binds to yeast yFEN-1. Either His6 or His6–yFEN-1 bound to the metal chelate resin was incubated with the cell extracts of transformed dna2-1 cells expressing WRN or WRN940–1432 and bound proteins that were eluted from the washed beads were resolved on 10% SDS–PAGE. WRN or WRN940–1432 was detected by western blot analysis using anti-WRN antibody and the resin bound His6–yFEN-1 was detected by anti-His6X antibody. Lane 1, input extract protein (25%) from dna2-z1/WRN; lane 2, input (10%) extract protein (25%) from dna2-1/WRN940–1432; lane 3, dna2-1/vector extract protein bound to His6–yFEN-1 resin; lane 4, dna2-1/WRN extract protein bound to His6–yFEN-1 resin; lane 5, dna2-1/WRN extract protein bound to His6 resin; lane 6, dna2-1/vector extract protein bound to His6 resin; lane 7, dna2-1/WRN940–1432 extract protein bound to His6 resin; lane 8, dna2-1/WRN940–1432 extract protein bound to His6–yFEN-1 resin. (B) WRN is associated with yFEN-1 in the transformed dna2-1 cells. Extracts from dna2-1 cells expressing WRN or WRN940–1432, yFEN-1–myc or both were prepared and subjected to immunoprecipitation (IP) with either anti-WRN or anti-myc antibodies, as indicated. Immunoprecipitated WRN or yFEN-1–myc were detected upon immunoblotting (IB) with anti-WRN or anti-myc antibodies, as indicated. Input signal for the yFEN-1–myc in the whole cell extracts of the transformed dna2-1 cells is shown (upper panel). (C and D) The C-terminal domain of WRN or BLM binds to yeast or human FEN-1 with similar affinity. Either BSA or purified recombinant human or yeast FEN-1 (23 nM) was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with serial dilutions (0–50 nM) of purified recombinant human WRN, WRN940–1432, BLM or BLM966–1417 for 1 h at 30°C. Following washing, FEN-1 bound WRN/WRN940–1432 or BLM/BLM966–1417 was detected by ELISA using rabbit polyclonal antibodies against WRN and BLM, respectively. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. (C) WRN or WRN940–1432 binding to hFEN-1 or yFEN-1. (D) BLM or BLM966–1417 binding to hFEN-1 or yFEN-1.

 
We performed co-immunoprecipitation experiments to explore the in vivo association of WRN/WRN940–1432 with yFEN-1. Yeast cells were co-transformed with the WRN or WRN940–1432 expression vectors and/or a myc-epitope-tagged yFEN-1 expression construct. An antibody against either the myc epitope or C-terminal epitope of WRN was used for the immunoprecipitation experiments from the yeast whole cell extracts. The anti-myc antibody precipitated yeast expressed WRN when yFEN-1–myc was also expressed (Fig. 7B). In control experiments, WRN was not precipitated by the anti-myc antibody from extracts of cells that did not harbor the yFEN-1–myc expression vector (Fig. 7B). The anti-WRN antibody precipitated yFEN-1–myc when WRN was also expressed, but did not precipitate yFEN-1–myc from extracts of cells lacking the WRN expression construct (Fig. 7B). Similarly, WRN940–1432 and yFEN-1–myc reciprocally co-immunoprecipitated with each other from extracts of yeast cells expressing both proteins (Fig. 7B). In control experiments, WRN940–1432 was not precipitated by the anti-myc antibody from extracts of cells that did not harbor the yFEN-1–myc plasmid, and yFEN-1–myc was not precipitated by anti-WRN antibody from extracts of cells lacking the WRN940–1432 expression vector (data not shown). The reciprocal co-immunoprecipitation of yFEN-1 and WRN or WRN940–1432 suggests that the proteins are associated with each other in a complex in vivo.

The C-terminal domain of WRN or BLM binds to yeast or human FEN-1 with similar affinity
To evaluate the specificity and nature of the WRN–yFEN-1 physical interaction, ELISA studies were performed. The fraction of WRN bound to yFEN-1 was both dose-dependent and saturable (Fig. 7C). The binding efficiency of WRN to yFEN-1 was comparable to hFEN-1. Specific binding of WRN to yFEN-1 or hFEN-1 was analyzed according to Scatchard binding theory by Hill plots. The apparent dissociation constants (Kd) for WRN–yFEN-1 and WRN–hFEN-1 were 2.00 and 1.34 nM, respectively (Table 1). These results indicate that WRN binds to yFEN-1 with similar affinity to that observed with the WRN–hFEN-1 interaction.


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  		Table 1. Apparent dissociation constants (Kd) for interaction between WRN or BLM with FEN-1
 
The ability of WRN940–1432 to rescue the dna2-1 mutant phenotypes in a similar fashion to what was observed for full-length WRN suggested that this non-catalytic C-terminal domain of WRN would interact with yFEN-1. WRN940–1432 binds to both yFEN-1 and hFEN-1 with similar affinity as evidenced by the calculated Kd values (Table 1). The physical analysis further demonstrated that WRN and WRN940–1432 bind with similar affinity to either yFEN-1 or hFEN-1 (Fig. 7C; Table 1).

On the basis of the functional interaction between BLM and yFEN-1 and the ability of a catalytically active BLM helicase to complement the dna2-1 cellular mutant phenotypes (34), we investigated the putative direct physical interaction between BLM and yFEN-1 by ELISA. The fraction of BLM bound to yFEN-1 was both dose-dependent and saturable (Fig. 7D). Thus BLM directly binds to yFEN-1 and with a similar affinity to that of the WRN–yFEN-1 interaction (Table 1). The BLM–yFEN-1 interaction was also determined to be of similar affinity to that of the BLM–hFEN-1 interaction (Table 1). BLM966–1417 binds to both yFEN-1 and hFEN-1 with similar affinity as observed for full-length BLM, full-length WRN or WRN940–1432. These results provide the first evidence that the non-catalytic C-terminal domain of BLM directly interacts with yFEN-1 or hFEN-1 with equal affinity to that of the WRN–FEN-1 interaction, suggesting a conserved function of a non-catalytic domain of BLM and WRN with FEN-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The critical roles of RecQ helicases to maintain genomic integrity are thought to derive from their ability to suppress recombination (40,41); however, the precise mechanisms are not well understood. Mutations in the WRN and BLM helicases are associated with a number of replication defects that include impaired fork progression, accumulation of abnormal replication intermediates and aberrant homologous recombination (HR). Evidence that replication forks of presumably damaged chromosomes stall normally in vivo suggests that cells are likely to have mechanisms to resolve abnormal DNA structures. At least one function of the WRN or BLM DNA helicases may be to prevent aberrant deleterious recombinogenic pathways when replication is perturbed by DNA damage, alternate DNA structure or impaired DNA synthesis.

Although the physical and functional interactions between WRN and FEN-1 have been characterized in vitro, the in vivo significance of this interaction was not clear. We hypothesized that the WRN–FEN-1 interaction might be important in vivo when DNA processing is defective during DNA replication. The fact that human and yeast FEN-1 are functionally conserved provided us an excellent opportunity to study the cellular functions of WRN in a defined genetic background. To simulate a condition of replicational stress, we chose to evaluate WRN function in a yeast dna2 mutant background characterized by a replication intermediate processing defect. In this study, we report that WRN complements the ts growth defect of a dna2-1 mutant. Analysis of DNA content by flow cytometry demonstrated that WRN/WRN940–1432 suppressed the dna2-1 mutant arrest with replicated DNA. The reduction in percentage of large-budded cells at the restrictive temperature by expression of WRN provided further evidence for the reversal of the G2/M arrest phenotype. Collectively, these results indicate that WRN compensates for Dna2 functions during late S phase to overcome G2/M arrest.

It was recently reported that expression of human BLM also complemented the dna2-1 ts growth phenotype (34). Mutant dna2-1 cells transformed with the same construct used to drive BLM expression but containing open reading frames encoding mouse WRN or human Rothmund–Thomson RecQ helicases remained ts for growth (34), suggesting that BLM might be unique in overcoming the dna2-1 replication defect. However, the low expression of either mouse WRN or human Rothmund–Thomson proteins in the transformed dna2-1 mutant cells did not permit the authors to draw any conclusions about the abilities of either mammalian RecQ helicase to suppress dna2-1 replication defects (34). The results presented in this study demonstrate that human WRN protein, expressed under gal induction from a multi-copy 2 µ plasmid, complemented the dna2-1 replication phenotypes. These findings suggest that WRN and BLM may act in the same cellular pathway(s) and that possibly a common domain of the two RecQ helicases may interact with the same DNA substrates and/or protein partners in vivo. As overexpression of yFEN-1 has been shown to genetically suppress dna2-1 defects in yeast, up-regulation of yFEN-1 cleavage activity by a number of mechanisms may suppress the dna2-1 mutation. Our yeast genetic results indicate that lower expression levels of WRN or WRN940–1432 when compared with the fully induced state at 2% gal were able to partially rescue the dna2-1 mutant phenotypes, suggesting that genetic complementation does not require gross WRN/WRN940–1432 overexpression and may be physiologically relevant. Extrapolation of the yeast genetic complementation data to human cells raises the question if WRN, BLM, one of the other human RecQ helicases, or another FEN-1 interactor such as PCNA might be the relevant player in vivo. The importance of the FEN-1–PCNA interaction for DNA repair processes has been shown in mammalian cells that conditionally express wild-type or defective mutant FEN-1 (42). A regulatable expression system for WRN or BLM in human cells will be required to delineate the physiological functions of human RecQ catalytic activities and protein interactions with DNA replication/repair factors including FEN-1 in genome stability maintenance.

It was conceivable that the RecQ helicase core domain was solely responsible for the dna2-1 complementation by WRN or BLM. The notion that helicase activity of BLM or WRN is essential for genetic rescue of the dna2 mutant would be consistent with the observation that the BLM-K695T ATPase mutant failed to complement the dna2-1 cellular phenotypes (34). However, other regions of WRN and BLM might be required for genetic rescue. Our previous work that mapped the minimal FEN-1 interaction domain of WRN to a region of the protein harboring the RQC motif (16) led us to hypothesize that this domain of WRN or BLM was necessary for complementation of the dna2-1 mutant cellular phenotypes. To address this possibility, we examined the ts phenotypes of a dna2-1 mutant that had been transformed with a plasmid encoding the C-terminal 940–1432 fragment of WRN that was previously shown to physically and functionally interact with hFEN-1. Our results indicate that this WRN domain was able to complement the ts lethality and MMS or HU sensitivity of dna2-1. Although both Dna2 and WRN have dual nuclease and helicase functions, the complementation was not due to a direct replacement of Dna2 functions by WRN because the cellular defects were alleviated by the non-catalytic WRN C-terminal domain alone. Importantly, this is the first evidence that a conserved non-catalytic protein domain fragment of a human RecQ helicase can function independently of the protein's catalytic activity in vivo.

The interaction between BLM or WRN helicases and FEN-1 supports the idea that either RecQ helicase may interact with endogenous yeast FEN-1 to rescue the dna2-1 mutant phenotype. We have demonstrated here that yFEN-1 cleavage of its preferred double-flap DNA substrates is stimulated by WRN940–1432 or BLM966–1417, indicating that catalytic activity of either human RecQ helicases is not necessary for the functional interaction in vitro. Both WRN and BLM bind to yFEN-1 with similar affinity compared to hFEN-1, suggesting that the RecQ–FEN-1 interaction is conserved among the species and is mediated by a conserved C-terminal domain of the human RecQ helicases WRN and BLM. The crystal structure of the conserved catalytic core of the E. coli RecQ helicase revealed that it comprises four independently folded structural subdomains: two of them form the helicase region and the two RQC subdomains constitute a Zn2+ binding domain and winged helix (43). Interestingly, the winged helix sub-domain aligns with the first 110 residues of the FEN-1 interaction domain of WRN (WRN949–1092) as well as a segment of the BLM966–1470 domain fragment that mediates the FEN-1 interaction (16). Thus, analysis of the related E. coli RecQ helicase core crystal structure suggests that the FEN-1 interaction domain of WRN or BLM folds independently from other regions of the protein and may be capable of functioning independently.

Although a non-catalytic domain of WRN or BLM protein is responsible for the FEN-1 interaction, genetic evidence indicates that helicase activity is also likely to be important in vivo. Cell cycle analyses of WRN transformed dna2-1 cells point to the possibility that WRN catalytic activity may be required for proper S-phase progression, but not essential for preventing the G2 delay suggesting that WRN may have enzymatic roles during DNA synthesis and non-enzymatic roles in checkpoint activation. The yeast-based WRN genetic complementation system can be further used for a WRN structure–function study to identify and characterize potentially separable functions of discrete WRN domains. A related approach in human cells was used recently to identify the roles of WRN biochemical activities (helicase, exonuclease) in cell survival and HR after DNA damage (44). In that work, it was shown that either WRN helicase or exonuclease activity was able to promote cell survival after DNA damage in the absence of recombination. The results from this study indicate that a non-catalytic domain of WRN can promote survival of the yeast dna2-1 mutant upon shift to restrictive temperature or exposure to MMS or HU.

In human cells, the phosphorylation status of FEN-1 or its interacting partner may regulate its role in replication or cell cycle progression. Phosphorylation of human FEN-1 by cyclin-dependent kinase Cdk1-Cyclin A in late S phase abrogates the interaction of FEN-1 with PCNA, preventing the stimulation of FEN-1 by PCNA (45). Phosphorylation of FEN-1 or WRN (46–49) may regulate the WRN–FEN-1 interaction and their coordinate action in S-phase progression, perhaps when the replication fork encounters DNA damage, or in the G2/M checkpoint.

Characterization of BS cell lines that had been stably transfected with BLM alleles encoding wild-type or mutated BLM protein with a K695T substitution in helicase motif I that inactivates ATPase/helicase activity demonstrated that BLM catalytic activity is necessary for correction of the elevated sister chromatid exchange of BS cells (50). In addition, most of the human cells transfected with the BLM-K695T allele died in culture, suggesting that this particular mutation may exert a dominant negative effect by the incorporation of BLM molecules that are inactive but of normal conformation to form poisonous complexes (50). Consistent with this notion, BLM-K695T was found to co-immunoprecipitate with yFEN-1 (34), suggesting that mutant BLM protein was associated with the replication fork complex; however, the catalytically inactive BLM-K695T mutant protein failed to complement the dna2-1 mutant (34). The results from this study indicate that the non-catalytic domain of WRN complemented dna2-1 cellular phenotypes, suggesting possible differences in complementation. Consistent with the notion that WRN and BLM may operate by different mechanisms in yeast, BLM helicase suppressed the cell growth in the top3 sgs1 mutation background and complemented the increased sensitivity of the sgs1 mutant to HU, but the WRN helicase did not (51).

The inactivation of biological function by the BLM K695T mutation was evidenced in yeast by the failure of the mutant allele to complement the HU-sensitive phenotype of a sgs1 top3 mutant (50) or the dna2-1 replication and repair phenotypes (34). Although these findings indicate the importance of BLM catalytic function in vivo, the functional significance of the BLM C-terminal domain was suggested by the observation that BS cells transfected with a BLM deletion allele in the RQC motif displayed genomic instability and defective nucleolar localization of the mutated BLM protein (52).

The biological importance of WRN catalytic activities (helicase, exonuclease) and WRN protein interactions in cellular DNA metabolism has been of considerable interest. Analysis of cell survival and HR in WS cell lines transfected with WRN exonuclease or helicase dead mutants that have been exposed to DNA damaging agents demonstrated that both catalytic activities were required for WRN to function in HR; however, the WRN mutant lacking either helicase or exonuclease activity enabled the cells to survive after DNA damage exposure in the absence of HR (44). These findings support a role for WRN catalytic function in vivo and are consistent with the absence of a single catalytic missense mutation in WS patients. In another study, WRN catalytic mutants were tested for their abilities to function in pathways of double-strand break repair in vivo and a structural role for WRN independent of its enzymatic activities was suggested to be important in optimizing HR and efficient non-homologous end-joining repair (53). We suggested a model in which WRN catalytic branch migration and physical/functional interaction with FEN-1 function together to process DNA structures associated with the regressed replication fork (30). Precisely at what stage WRN is acting in processing replication fork structures after DNA damage is not known. The ability of the WRN non-catalytic domain to complement the dna2-1 hypersensitivity to HU and MMS suggests that in addition to its catalytic functions, a role for WRN protein interaction(s) in processing certain DNA replication or recombination intermediates is important.

RecQ mutants are sensitive to HU (40,41), an agent that leads to S-phase arrest with stalled replication forks. WS cells display a reduced rate of repair, elevated apoptotic cell death and increased DNA strand breaks after replication arrest by HU treatment or DNA damage induced in S phase (6). The observation that expression of a Holliday Junction resolvase rescues both the recombination defect and cell survival following DNA damage in WS cells (54) suggests that inappropriate processing of stalled replication fork intermediates directly contributes to the aberrant HR characteristic of WS cells. Moreover, suppression of RAD51-dependent recombination significantly improved survival of WS cells following DNA damage (54).

In human cells, WRN and FEN-1 form a complex upon replication arrest (30), suggesting that WRN and FEN-1 together might facilitate processing of certain DNA structures that interfere with normal DNA transactions when replication is perturbed. The replication defects in WS cell lines point to an underlying deficiency when fork progression is abnormal. Co-localization of WRN with arrested replication forks in response to HU treatment (55) or DNA damage (30) is consistent with its role in response to replicational stress. Asymmetry of DNA replication fork progression in WS cells suggests that WRN acts to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks and that loss of this capacity may be a contributory factor in premature ageing (56). The remarkable ability of WRN to complement the HU sensitivity of a dna2-1 mutant further demonstrates an important role of WRN in a pathway that deals with stalled replication. Mutations in DNA replication genes (dna2, rad27) reduce yeast life span (57) and the shortened lifespan is proposed to be at least partly due to increased replication fork stalling and/or defects in repair of stalled forks leading to hyperrecombination and chromosome instability (58). Collectively, the results presented here and published work (30,32,37) suggest that either BLM or WRN participate with FEN-1 in the process of DNA replication to insure the fidelity of genome maintenance, perhaps under specialized circumstances such as when the replication fork encounters a block.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cloning
Coding sequence of either full-length human WRN or WRN940–1432 was PCR amplified from a WRN cDNA plasmid kindly provided by Dr J. Oshima (University of Washington Medical School, Seattle, WA, USA). PCR products were subcloned into the SalI–MluI sites of vector YEp195SpGAL (17), a 2 µm multi-copy plasmid containing a URA3 selectable marker, to construct YEp195SpGAL–WRN or YEp195SpGAL–WRN940–1432, which express full-length WRN or WRN940–1432, respectively, under the control of a gal-inducible promotor. The expression plasmids pRG106A derived from the vector YEp195SpGAL for gal-inducible overexpression of wild-type RAD27 in yeast cells and pLC76A derived from the pET28 vector for isoplopyl-ß-D-thiogalactoside (IPTG)-inducible expression of wild-type RAD27 with C-terminal His6 tag in bacterial cells were kindly provided by Dr R. Gary (University of Nevada, Las Vegas, NV, USA). The yeast expression plasmid pJDG–Rad27–myc encoding yFEN-1–myc was generously provided by Dr J. Campbell (California Institute of Technology, Pasadena, CA, USA).

Media and strains
Strains with wild-type DNA2 (WT; 4053-5-2, genotype MATa trp1 leu2 ura3 his7) or a dna2-1 missense allele (integrated into the genome; genotype MATa trp1 leu2 ura3 his7 can1 dna2-1) have been characterized (35) and were a gift from Dr T. Formosa (University of Utah, Salt Lake City, UT, USA). Yeast cultures were grown using standard protocol and transformations were performed using Yeastmaker yeast transformation system 2 (BD Biosciences). Transformed yeast strains were grown in SC media containing 2% raffinose minus uracil (Ura). Two percent raffinose was included in SC media containing gal.

Western blot analyses
Transformed dna2-1 strains were grown in SC raffinose minus Ura at 23°C to an OD600 of 0.5 and WRN expression was induced by the indicated concentration of gal. Following the indicated time of gal induction, cells (0.5 ml) were collected by centrifugation, washed with phosphate-buffered saline (PBS), lysed in alkaline lysis buffer [50 mM NaOH, pH 10.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and protease inhibitors (Roche Molecular Biochemicals)], boiled for 5 min, clarified by centrifugation and neutralized with 1 M HCl. Proteins from equivalent amounts of cell lysate were resolved on 10% polyacrylamide SDS gels. Expression of WRN or WRN940–1432 was determined by western blot using a WRN monoclonal antibody directed against an epitope in the WRN C-terminus (1 : 250, BD Transduction Labs).

Genetic complementation assays
dna2-1 cells transformed with YEp195SpGAL, YEp195SpGAL–WRN, YEp195SpGAL–WRN940–1432 or YEp195SpGAL–RAD27 were grown in SC raffinose minus Ura at 23°C. Overnight cultures were diluted to an OD600 of 0.2 and grown for an additional 4–5 h. Ten-fold serial dilutions of these strains were spotted onto SC glucose or SC gal minus Ura plates with or without MMS (0.006%) or HU (150 mM). Plates were incubated for 5–7 days at 23°C. Control plates were incubated for 3–5 days. For temperature-sensitive studies, transformed yeast strains were streaked onto SC gal minus Ura plates and incubated for 3–5 days at 23°C or 5–7 days at 37°C.

Flow cytometry and DAPI staining
Transformed yeast strains were grown in SC raffinose minus Ura to ~1x107 cells/ml at 23°C. Cultures were split, spun down, resuspended in prewarmed (37°C) or room temperature SC gal minus Ura media and incubated for 4 h at 37 or 23°C, respectively. Cells (5–10 ml) were sonicated for 20 s on ice with a microtip sonicator (Microson Ultrasonic Cell Disruptor, setting 2), washed once with Tris buffer (50 mM Tris–HCl, pH 7.5, 15 mM NaCl), fixed in 70% ethanol overnight at 4°C, processed for Sytox green staining for flow cytometric analysis (59) and analyzed on a FACScan (Becton Dickinson). Ten thousand events were analyzed for each sample.

For DAPI (4', 6-diamidino-2-phenylindole) staining, 70% ethanol fixed cells were washed with 1x PBS, stained and mounted in Vectashield mounting medium with DAPI (1 µg/ml) (Vector, USA). Cells were examined with an Axiovert 200 M microscope (Zeiss; 40x lens) and composite differential interference contrast (DIC) and fluorescence images were analyzed using AxioVision, version 3.0 program (Zeiss).

Recombinant proteins
Human His6–WRN protein was overexpressed using a baculovirus/Sf9 insect system and purified as described (30). Human His6–WRN940–1432, overexpressed in E. coli, was purified as published (60). Human His6–BLM protein (61) and BLM966–1417, fused to MBP (62), were kindly provided by Dr I. Hickson (Cancer Research UK Laboratories). His6-tagged yFEN-1, kindly provided by Dr R. Bambara (Department of Biochemistry and Biophysics, University of Rochester Medical Center), and hFEN-1 were overexpressed in E. coli and purified as described (30,37).

Oligonucleotide substrates
PAGE-purified oligonucleotides (Midland Certified Reagent Co.) were used for DNA substrate preparation. Double-flap substrates with either a 1 nt 3' complementary tail or an equilibrating 12 nt 3' tail were constructed as described using D1 : T1: U2 and D2 : T1 : U8, respectively (37).

FEN-1 incision assays
Reactions (20 µl) contained 10 fmol DNA substrate and the specified amounts of recombinant BLM, BLM966–1417, WRN or WRN940–1432 and/or human or yeast FEN-1 in 30 mM HEPES (pH 7.6), 5% glycerol, 40 mM KCl, 0.1 mg/ml bovine serum albumin (BSA) and 8 mM MgCl2. BLM or WRN was mixed with the substrate and buffer on ice prior to the addition of FEN-1. Reactions were incubated at 37°C for 15 min, terminated by the addition of 10 µl of formamide stop solution [80% formamide (v/v), 0.1% bromophenol blue and 0.1% xylene cyanole] and then heated to 95°C for 5 min. Products were resolved on 20% polyacrylamide, 7 M urea denaturing gels and quantitated as described (16). Cleavage data represent the mean of at least three independent experiments with standard deviations shown by error bars.

Yeast yFEN-1 affinity pull-down experiments
His6-tagged yFEN-1 or His6 alone were overexpressed in bacteria as previously described (63). The bacterial cell pellet was lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl and 0.2 mg/ml lysozyme) supplemented with protease inhibitors (Roche Molecular Biochemicals) and clarified at 16 000g for 30 min at 4°C. An aliquot of 1 ml of the resulting lysate was incubated with 100 µl of Ni2+ agarose beads (50% v/v, Qiagen) for 1 h at 4°C. The beads were washed three times with 1 ml of lysis buffer, followed by three washes with lysis buffer containing 60 mM imidazole. Typically, ~300–400 ng of yFEN-1 bound to the washed Ni2+ agarose beads was used for each affinity pull-down experiment. For preparation of yeast cell extracts, dna2-1 cells transformed with YEp195SpGAL, YEp195SpGAL–WRN or YEp195SpGAL–WRN940–1432 were grown in SC raffinose minus Ura at 23°C to an OD600 of 0.5 and WRN expression was induced by 2% gal. Following a 4 h induction, cells were pelleted and washed with sterile water, lysed in yeast lysis buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2% NP-40, 10% glycerol, 15 mM dithiothreitol (DTT) 1 mM PMSF and protease inhibitors (Roche Molecular Biochemicals)] with glass beads (Sigma) and clarified at 16 000g for 15 min at 4°C. For binding experiments, the His6 or His6–yFEN-1 bound beads were then incubated for 1 h at 4°C with yeast cell extract (250 µl) by gentle rotation at 4°C. The beads were subsequently washed three times with 500 µl of yeast lysis buffer and eluted by boiling treatment in 40 µl of Laemmli buffer. Proteins were electrophoresed on 10% polyacrylamide SDS gels and transferred to PVDF membranes. Control membranes were stained with amido black reagent to demonstrate equal protein loading for samples. Membranes were probed with mouse monoclonal anti-WRN antibody (1 : 250, BD Transduction Labs) or the rabbit polyclonal anti-6XHis (1 : 2000, Abcam) followed by horse anti-mouse IgG-horseradish peroxidase (HRP) (Vector) or donkey anti-rabbit IgG-HRP (Santa Cruz Biotech.), respectively, and ECL-Plus (Amersham Pharmacia).

Co-immunoprecipitation experiments
For preparation of yeast cell extracts, the transformed dna2-1 cells were grown in yeast extract/peptone/raffinose (YPR) at 30°C to an OD600 of 0.5 and the WRN or yFEN-1–myc expression was induced by 2% gal. Following a 4 h induction, cells were pelleted and washed twice with cold sterile water, lysed in yeast lysis buffer with glass beads (Sigma) and clarified at 16 000g for 15 min at 4°C. The supernatants were further diluted in yeast lysis buffer and incubated at 4°C for 2 h with anti-WRN (1 : 40, Santa Cruz Biotech) or anti-myc (1 : 40, Santa Cruz Biotech) antibodies followed by incubation with the protein G–agarose beads (Amersham) for 2 h at 4°C with gentle tumbling. The beads were washed four times with yeast lysis buffer supplemented with protease inhibitors and the immunoprecipitates were eluted by boiling in Laemmli buffer and resolved on 10% SDS–PAGE. Immunoprecipitated WRN and yFEN-1–myc were detected by western blotting using anti-WRN (BD Transduction) or anti-myc (Santa Cruz Biotech) antibodies.

ELISA
Microtiter plates were coated with 23 nM purified recombinant hFEN-1, yFEN-1 or BSA and ELISA was performed as described (39). The fraction of BLM/BLM966–1417 or WRN/WRN940–1432 that was bound by yFEN-1 or hFEN-1 was detected using rabbit anti-BLM (1 : 250, Abcam) or anti-WRN (1 : 500, Santa Cruz) antibodies. A Hill plot was used to analyze the data as described (64).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Drs L. Wu and I. Hickson (Cancer Research UK Laboratories) for recombinant BLM and BLM966–1417 and Drs Y. Liu and R. Bambara (Department of Biochemistry and Biophysics, University of Rochester Medical Center) for recombinant yFEN-1. We acknowledge Drs D. Gordenin and F. Storici (NIEHS-NIH) for advice on WRN cloning into yeast expression vector. We thank Dr T. Formosa (University of Utah) for dna2 strains, Dr J. Campbell (California Institute of Technology) for a yeast expression plasmid encoding yFEN-1 with a myc epitope, Dr R. Gary (UNLV) for the RAD27 expression plasmids and NIA IRP Flow Cytometry Laboratory (NIH) (Dr R. Wersto, F.J. Chrest and C. Morris) for assistance with FACS analysis. We thank members of the Laboratory of Molecular Gerontology (NIA-NIH) for helpful discussion and Drs K. Doherty and D. Wilson III (NIA-NIH) for critical reading of the manuscript. This work was funded in part by grant GM24441 from NIH.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +14105588578; Fax: +1 4105588157; Email: broshR{at}grc.nia.nih.gov


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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