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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.

Aggarwal M, Brosh RM - Aging (Albany NY) (2009)

Bottom Line: 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.Proposed roles of WRN in genetic pathways important for the suppression of genomic instability are discussed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, NIH, NIH Biomedical Research Center, 251 Bayview Blvd, Suite 100, Rm #06B125, Baltimore, MD 21224, USA.

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.

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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.
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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.

Mentions: Previously, it was reported that the sgs1 top3 double mutant is less sensitive to MMS or HU than the top3 single mutant [6]. Since WRN affected cell growth of sgs1 top3, we next examined its effect on drug sensitivity. As shown in Figure 6, sgs1 top3/WRN displayed sensitivity to both MMS and HU that was comparable to sgs1 top3/SGS1, whereas sgs1 top3/vector was more resistant to either drug. Genetic analysis of the WRN variants in the sgs1 top3 background revealed that strains transformed with WRN-K577M, WRN-K1016A, or WRN-R834C displayed sensitivity comparable to that of vector, whereas the sgs1 top3/WRN-E84A strain showed HU and MMS resistance similar to sgs1 top/WRN and sgs1 top3/SGS1 (Figure 6). These results demonstrate that in addition to its effect on growth in the sgs1 top3 background, WRN also affects sensitivity of the sgs1 top3 mutant to HU or MMS. Furthermore, WRN ATPase/helicase but not exonuclease activity is genetically required for its effect on HU or MMS sensitivity in the sgs1 top3 background. Expression of WRN in the wild-type parent strain had no effect on the strain's sensitivity to the tested concentration of HU or MMS (Supp. Data Figure 4), indicating that the effect of WRN expression was dependent on the sgs1 top3 genetic background.


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.

Aggarwal M, Brosh RM - Aging (Albany NY) (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: Previously, it was reported that the sgs1 top3 double mutant is less sensitive to MMS or HU than the top3 single mutant [6]. Since WRN affected cell growth of sgs1 top3, we next examined its effect on drug sensitivity. As shown in Figure 6, sgs1 top3/WRN displayed sensitivity to both MMS and HU that was comparable to sgs1 top3/SGS1, whereas sgs1 top3/vector was more resistant to either drug. Genetic analysis of the WRN variants in the sgs1 top3 background revealed that strains transformed with WRN-K577M, WRN-K1016A, or WRN-R834C displayed sensitivity comparable to that of vector, whereas the sgs1 top3/WRN-E84A strain showed HU and MMS resistance similar to sgs1 top/WRN and sgs1 top3/SGS1 (Figure 6). These results demonstrate that in addition to its effect on growth in the sgs1 top3 background, WRN also affects sensitivity of the sgs1 top3 mutant to HU or MMS. Furthermore, WRN ATPase/helicase but not exonuclease activity is genetically required for its effect on HU or MMS sensitivity in the sgs1 top3 background. Expression of WRN in the wild-type parent strain had no effect on the strain's sensitivity to the tested concentration of HU or MMS (Supp. Data Figure 4), indicating that the effect of WRN expression was dependent on the sgs1 top3 genetic background.

Bottom Line: 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.Proposed roles of WRN in genetic pathways important for the suppression of genomic instability are discussed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, NIH, NIH Biomedical Research Center, 251 Bayview Blvd, Suite 100, Rm #06B125, Baltimore, MD 21224, USA.

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.

Show MeSH
Related in: MedlinePlus