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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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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 OD0.05                                            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.
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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 OD0.05 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.

Mentions: sgs1 top3 double mutant cells transformed with YEp112SpGAL or YEp112SpGAL-WRN were streaked onto plates containing SC minus Trp media with 2% gal. As shown in Figure 2A, the WRN transformed sgs1 top3 strain grew significantly slowly compared to the vector transformed sgs1 top3 strain. sgs1 top3 transformed with the YEp112SpGAL-SGS1 plasmid was included as a positive control (Figure 2A), demonstrating that wild-type Sgs1 expressed in the sgs1 top3 mutant was able to genetically complement the growth phenotype. The transformed sgs1 top3 strains grew similarly in the absence of gal as shown for 2 days (Figure 2A) or earlier time points (data not shown). Expression of WRN had no effect on the growth of parental wild-type (W3031A) or sgs1 strains (Figure 2B), indicating that the effect of WRN on cell growth was specific to that observed in the sgs1 top3 mutant 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)

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 OD0.05                                            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.
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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 OD0.05 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.
Mentions: sgs1 top3 double mutant cells transformed with YEp112SpGAL or YEp112SpGAL-WRN were streaked onto plates containing SC minus Trp media with 2% gal. As shown in Figure 2A, the WRN transformed sgs1 top3 strain grew significantly slowly compared to the vector transformed sgs1 top3 strain. sgs1 top3 transformed with the YEp112SpGAL-SGS1 plasmid was included as a positive control (Figure 2A), demonstrating that wild-type Sgs1 expressed in the sgs1 top3 mutant was able to genetically complement the growth phenotype. The transformed sgs1 top3 strains grew similarly in the absence of gal as shown for 2 days (Figure 2A) or earlier time points (data not shown). Expression of WRN had no effect on the growth of parental wild-type (W3031A) or sgs1 strains (Figure 2B), indicating that the effect of WRN on cell growth was specific to that observed in the sgs1 top3 mutant 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