WRN helicase defective in the premature aging disorder Werner syndrome genetically interacts with topoisomerase 3 and restores the top3 slow growth phenotype of sgs1 top3

Abstract

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability. The WRN gene defective in WS encodes a protein with both helicase and exonuclease activities that interacts with proteins implicated in DNA metabolism. To understand its genetic functions, we examined the ability of human WRN to rescue phenotypes associated with sgs1, the sole RecQ helicase in Saccharomyces cerevisiae. WRN failed to rescue sgs1 sensitivity to the DNA damaging agent methylmethane sulfonate or replication inhibitor hydroxyurea, suggesting divergent functions of human and yeast RecQ helicases. However, physiological expression of WRN in sgs1 top3 restored top3 slow growth phenotype, whereas no effect on growth was observed with wild-type or sgs1 strains. Slow growth of WRN-transformed sgs1 top3 correlated with an elevated population of large-budded cells with undivided nuclei, indicating restoration of cell cycle delay in late S/G2 characteristic of top3. WRN helicase but not exonuclease activity was genetically required for restoration of top3 growth phenotype, demonstrating separation of function of WRN catalytic activities. A naturally occurring missense polymorphism in WRN that interferes with helicase activity abolished its ability to restore top3 slow growth phenotype. Proposed roles of WRN in genetic pathways important for the suppression of genomic instability are discussed.

Keywords: Bloom's syndrome; RecQ; Sgs1; Werner syndrome; genomic instability; helicase; topoisomerase.

Figure 1.. WRN fails to rescue the MMS and HU sensitivity of sgs1.

Cultures of wild-type parental strain (W303-1A) or sgs1 strain transformed with YEp112SpGAL or YEp112SpGAL-WRN were grown to early log phase (OD ₆₀₀ of ~0.6 to 0.8). Ten-fold serial dilutions of these cultures were spotted onto SC-Trp plates containing 2% gal and either MMS or HU at the indicated concentrations. Plates were incubated at 30°C for 3 days (control plates) and 5 days (MMS or HU plates) and then photographed.

Figure 2.. WRN expression in sgs1 top3 restores the slow growth phenotype of top3.

Panel A, sgs1 top3 strain transformed with YEp112SpGAL or YEp112SpGAL-WRN were streaked on an SC-Trp plate containing either 2% glu or 2% gal. As a control sgs1 top3 strain transformed with YEp112SpGAL-SGS1 was streaked on both the plates. Plates were incubated at 30°C for 2 days and then photographed. Panel B, Wild type parental strain W303-1A or sgs1 strain transformed with YEp112SpGAL or YEp112SpGAL-WRN were streaked on an SC-Trp plate either containing 2% glu or 2% gal. Plates were incubated at 30°C for 4 days and then photographed. Composition of the plates was as in Panel A and Panel B respectively. Panel C, Comparison of growth of sgs1 top3 strain transformed with YEp112SpGAL, YEp112SpGAL-WRN or YEp112SpGAL-SGS1. Logarithmically growing cultures of above mentioned strains were reinoculated at OD_0.05 in SC-Trp medium containing 2% gal and were incubated at 30°C. Growth of the cultures was followed by their absorbance at OD₆₀₀. The experiment was repeated twice in duplicate with similar results. Data represent the mean with standard deviations indicated by error bars.

Figure 3.. WRN expression induces S/G2 arrest in sgs1 top3 cells.

Logarithmically growing cultures of sgs1 top3 strain transformed with YEp112SpGAL, YEp112SpGAL-WRN, or YEp112SpGAL-SGS1, and the vector-transformed wild-type parental strain were induced at 2% gal concentration for 6 h. Cultures were harvested, processed for DAPI staining as described in "Materials and Methods" and were observed using Axiovert 200 M microscope(Zeiss; 100x lens). Shown is the DAPI staining of the sgs1 top3 transformed with YEp112SpGAL (upper left) and with YEp112SpGAL-WRN (upper right). Arrows show cells with undivided nuclei. Distribution of the cells in G1 (single cells) and S/G2 (budded cells) is shown in lower panel.

Figure 4.. WRN ATPase/helicase, but not exonuclease activity, is required to restore the slow growth phenotype of top3 in sgs1 top3 background.

Panel A, WRN protein with conserved domains and positions of site-directed mutations. Panel B, Expression of WRN and WRN variants in transformed sgs1 top3 was induced at 2% gal concentration and cells were harvested after 6 h. Equal amount of total cell lysate was loaded on to 8-16% polyacrylamide SDS gels, followed by Western blotting using anti-WRN antibody. sgs1 top3 strain transformed with ATPase/helicase-dead (YEp195SpGAL-WRN K577M), exonuclease-dead (YEp195SpGAL-WRN E84A), RQC mutant (YEp195SpGAL-WRN K1016A), or polymorphic mutant (YEp195SpGAL-WRN R834C) was streaked on SC-Trp plates containing either 2% glu (Panel E) or 2% gal (Panel D). Plates were incubated at 30°C for 2 days and then photographed. Composition of the plates was as in Panel C.

Figure 5.. WRN mediated restoration of slow growth phenotype in sgs1 top3 background is independent of its expression level.

WRN expression in the transformed sgs1 top3 cells was induced with the indicated gal concentrations and cells were harvested 6 h after induction. As a control, sgs1 top3/YEp112SpGAL was included. Equal amounts of total cell lysate were loaded on 8-16% polyacrylamide SDS gels followed by Western blot detection using anti-WRN antibody as shown in Panel A. sgs1 top3 strain transformed with YEp112SpGAL, YEp112SpGAL-WRN, exonuclease-dead (YEp195SpGAL-WRN E84A), ATPase/helicase-dead (YEp195SpGAL-WRN K577M), RQC mutant (YEp195SpGAL-WRN K1016A), polymorphic mutant(YEp195SpGAL-WRN R834C) and YEp112SpGAL-SGS1 was streaked on SC-Trp plate containing gal at varying concentrations asindicated. Plates were incubated at 30°C for 2 days and then photographed. Panels C-F show the effect of WRN expression onthe growth rate of sgs1 top3 transformed strains at different gal concentrations. Composition of the plates was as in Panel B.

Figure 6.. Effect of WRN expression on the MMS and HU sensitivity of sgs1 top3 strain.

Logarithmically growing cultures of sgs1 top3 strain transformed with YEp112SpGAL, YEp112SpGAL-WRN, exonuclease-dead (YEp195SpGAL-WRN E84A), ATPase/helicase-dead (YEp195SpGAL-WRN K577M), RQC mutant (YEp195SpGAL-WRN K1016A), polymorphic mutant (YEp195SpGAL-WRN R834C), YEp112SpGAL-SGS1 and vector transformed wild type parental strains were spotted in a ten-fold serial dilutions onto SC-Trp plates containing glu or gal and either MMS or HU at the indicated concentrations. Plates were incubated at 30°C for 2 days (control plates) and 4 days (MMS and HU plates) and then photographed.