Limits...
Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response

Srividya I, Tirupataiah S, Mishra K - PLoS ONE (2012)

Bottom Line: Yku70/80 proteins are associated with telomeres and are important for maintaining the integrity of telomeres.One of the suppressors identified was RTT103, which encodes a protein implicated in transcription termination.We show that rtt103Δ are sensitive to multiple forms of genome insults and that RTT103 is essential for recovery from DNA double strand breaks in the chromosome.

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.

ABSTRACT

YKu70/YKu80 is a heterodimer that is essential for repair of DNA double strand breaks through non-homologous end joining pathway in the yeast Saccharomyces cerevisiae. Yku70/80 proteins are associated with telomeres and are important for maintaining the integrity of telomeres. These proteins protect telomeres from recombination events, nuclease attacks, support the formation of heterochromatin at telomeres and anchor telomeres to the nuclear periphery. To identify components in molecular networks involved in the multiple functions of Yku70/80 complex, we performed a genetic screen for suppressors of yku70 deletion. One of the suppressors identified was RTT103, which encodes a protein implicated in transcription termination. We show that rtt103Δ are sensitive to multiple forms of genome insults and that RTT103 is essential for recovery from DNA double strand breaks in the chromosome. We further show that Rtt103 associates with sites of DNA breaks and hence is likely to play a direct role in response to DNA damage.

RTT103 partially suppresses the the MMS sensitivity of yku70.WT (KRY193) and yku70 (KRY 172) mutants were transformed with empty vector, KM93 and RTT103. 5 µl of 10-fold serial dilutions of yeast cultures were plated on SC-LEU plates containing MMS and incubated at 30°C for 2–3 days. (2b) Quantification of MMS sensitivity. The MMS sensitivity of yku70 was quantified by plating appropriate dilutions on plates with and without MMS. Sensitivity of WT was set to 1. yku70 is approximately 60 fold sensitive and upon overexpression of RTT103 the sensitivity to MMS is reduced by approximately 8 fold. Quantification was done for three independent cultures and error bars show SD.
© Copyright Policy

pone-0031288-g002: RTT103 partially suppresses the the MMS sensitivity of yku70.WT (KRY193) and yku70 (KRY 172) mutants were transformed with empty vector, KM93 and RTT103. 5 µl of 10-fold serial dilutions of yeast cultures were plated on SC-LEU plates containing MMS and incubated at 30°C for 2–3 days. (2b) Quantification of MMS sensitivity. The MMS sensitivity of yku70 was quantified by plating appropriate dilutions on plates with and without MMS. Sensitivity of WT was set to 1. yku70 is approximately 60 fold sensitive and upon overexpression of RTT103 the sensitivity to MMS is reduced by approximately 8 fold. Quantification was done for three independent cultures and error bars show SD.

Mentions: Since YKU70 is involved in several processes including DNA repair, telomere metabolism and gene silencing we tested if multiple copies of RTT103 also suppressed any of these phenotypes. We found that while neither gene silencing nor telomere length defects of yku70 mutants were affected by RTT103 (data not shown), the DNA repair phenotype was partially suppressed by over-expression of RTT103 (Figure 2). yku70/80 mutants are sensitive to MMS, an alkylating agent, which produces DSB during repair. Therefore, we plated wild type and yku70Δ transformed with either empty vector or RTT103 on plates containing MMS. As shown in Figure 2a and Figure 2b, yku70 mutants are over 50-fold more sensitive to MMS than wild type (row 4). Upon elevated dosage of RTT103, there is a marked improvement in survival, and these cells are only 7-fold more sensitive than wild type, suggesting that RTT103 on multicopy plasmid partially suppresses the yku70 temperature sensitivity and MMS sensitivity. RTT103 on single copy plasmid could not suppress these phenotypes indicating that suppression requires multiple copies of RTT103 (data not shown).

View Similar Images In: Results  - Collection
View Article: PubMed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=3280293&rFormat=json&query=null&req=5
Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response

Srividya I, Tirupataiah S, Mishra K - PLoS ONE (2012)

RTT103 partially suppresses the the MMS sensitivity of yku70.WT (KRY193) and yku70 (KRY 172) mutants were transformed with empty vector, KM93 and RTT103. 5 µl of 10-fold serial dilutions of yeast cultures were plated on SC-LEU plates containing MMS and incubated at 30°C for 2–3 days. (2b) Quantification of MMS sensitivity. The MMS sensitivity of yku70 was quantified by plating appropriate dilutions on plates with and without MMS. Sensitivity of WT was set to 1. yku70 is approximately 60 fold sensitive and upon overexpression of RTT103 the sensitivity to MMS is reduced by approximately 8 fold. Quantification was done for three independent cultures and error bars show SD.
© Copyright Policy
pone-0031288-g002: RTT103 partially suppresses the the MMS sensitivity of yku70.WT (KRY193) and yku70 (KRY 172) mutants were transformed with empty vector, KM93 and RTT103. 5 µl of 10-fold serial dilutions of yeast cultures were plated on SC-LEU plates containing MMS and incubated at 30°C for 2–3 days. (2b) Quantification of MMS sensitivity. The MMS sensitivity of yku70 was quantified by plating appropriate dilutions on plates with and without MMS. Sensitivity of WT was set to 1. yku70 is approximately 60 fold sensitive and upon overexpression of RTT103 the sensitivity to MMS is reduced by approximately 8 fold. Quantification was done for three independent cultures and error bars show SD.
Mentions: Since YKU70 is involved in several processes including DNA repair, telomere metabolism and gene silencing we tested if multiple copies of RTT103 also suppressed any of these phenotypes. We found that while neither gene silencing nor telomere length defects of yku70 mutants were affected by RTT103 (data not shown), the DNA repair phenotype was partially suppressed by over-expression of RTT103 (Figure 2). yku70/80 mutants are sensitive to MMS, an alkylating agent, which produces DSB during repair. Therefore, we plated wild type and yku70Δ transformed with either empty vector or RTT103 on plates containing MMS. As shown in Figure 2a and Figure 2b, yku70 mutants are over 50-fold more sensitive to MMS than wild type (row 4). Upon elevated dosage of RTT103, there is a marked improvement in survival, and these cells are only 7-fold more sensitive than wild type, suggesting that RTT103 on multicopy plasmid partially suppresses the yku70 temperature sensitivity and MMS sensitivity. RTT103 on single copy plasmid could not suppress these phenotypes indicating that suppression requires multiple copies of RTT103 (data not shown).

Bottom Line: Yku70/80 proteins are associated with telomeres and are important for maintaining the integrity of telomeres.One of the suppressors identified was RTT103, which encodes a protein implicated in transcription termination.We show that rtt103Δ are sensitive to multiple forms of genome insults and that RTT103 is essential for recovery from DNA double strand breaks in the chromosome.

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.

ABSTRACT

YKu70/YKu80 is a heterodimer that is essential for repair of DNA double strand breaks through non-homologous end joining pathway in the yeast Saccharomyces cerevisiae. Yku70/80 proteins are associated with telomeres and are important for maintaining the integrity of telomeres. These proteins protect telomeres from recombination events, nuclease attacks, support the formation of heterochromatin at telomeres and anchor telomeres to the nuclear periphery. To identify components in molecular networks involved in the multiple functions of Yku70/80 complex, we performed a genetic screen for suppressors of yku70 deletion. One of the suppressors identified was RTT103, which encodes a protein implicated in transcription termination. We show that rtt103Δ are sensitive to multiple forms of genome insults and that RTT103 is essential for recovery from DNA double strand breaks in the chromosome. We further show that Rtt103 associates with sites of DNA breaks and hence is likely to play a direct role in response to DNA damage.

View Similar Images In: Results  - Collection
View Article: PubMed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=3280293&rFormat=json&query=null&req=5