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RTT103 partially suppresses the yku70 ts phenotype.WT (KRY193) and yku70 (KRY172) 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 and incubated at 30°C, 35°C and 37°C for 2–3 days. (1b) Quantification of temperature sensitivity. The temperature sensitivity of yku70 was quantified by plating out appropriate dilutions of 3 independent cultures at the appropriate temperatures. Sensitivity of WT was set to 1. yku70 is approximately 40 fold sensitive and upon overexpression of RTT103, the sensitivity to temperature is reduced by approximately 7 to 8 fold. Error bars indicate SD.
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pone-0031288-g001: RTT103 partially suppresses the yku70 ts phenotype.WT (KRY193) and yku70 (KRY172) 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 and incubated at 30°C, 35°C and 37°C for 2–3 days. (1b) Quantification of temperature sensitivity. The temperature sensitivity of yku70 was quantified by plating out appropriate dilutions of 3 independent cultures at the appropriate temperatures. Sensitivity of WT was set to 1. yku70 is approximately 40 fold sensitive and upon overexpression of RTT103, the sensitivity to temperature is reduced by approximately 7 to 8 fold. Error bars indicate SD.

Mentions: Two plasmids that reproducibly improved growth were obtained in this screen; one of the plasmids (KM95), showed strong suppression of yku70 phenotype and upon sequencing was found to contain the full length YKU70 gene. This YKU70 gene complemented the yku70 phenotype (not shown). The second plasmid (KM93) that partially suppressed the yku70 temperature sensitivity phenotype contained the complete sequence of the RTT103 gene. RTT103 was subcloned in YEplac181 vector and was tested to see if it suppressed yku70 phenotype (Figure 1a). As shown in Figure 1a, yku70Δ is lethal at 37°C and partially defective for growth at 35°C. Upon over-expression of KM93 or just RTT103 on a multicopy plasmid, yku70Δ grew better at 35°C (compare row 4 with rows 5 and 6). As extent of suppression of temperature sensitivity was modest, we quantified the phenotype by plating cells at different temperatures. As shown in Figure 1b, we found that elevated dosage of RTT103 improved growth of yku70Δ by 7 to 8-fold. However, RTT103 could not suppress lethality at 37°C. Therefore, we conclude that an elevated dose of RTT103 partially suppresses the temperature sensitivity of yku70 mutants.

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.

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