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Results: 8

1.
Figure 7

Figure 7. rtt103Δ do not show hyper-recombination phenotype.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

(7a) Schematic representation of chromosomal region containing leu2-k repeats flanking the ADE2 and URA3 genes. (7b) WT (KRY615), rtt103 (KRY650), hpr1 (KRY652), strains containing the ADE2, URA3 genes flanked by leu2-k repeats were plated on SC plates (viability) and SC+FOA plates (recombinants). Recombination between the leu2 repeats leads to loss of ADE2 and URA3 genes. The recombination frequency was determined for three independent segregants and error bars denote SD.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
2.
Figure 6

Figure 6. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

(6a) rat1-1 and rai1 mutants are not sensitive to MMS. WT (KRY105), rtt103 (KRY230) WT RAI1 (KRY631), rai1 (KRY632), WT RAT1 (KRY633) and rat1-1 (KRY634) strains were grown to mid-log phase. 5 µl of 10-fold serial dilutions of yeast cultures were plated on YPD plates containing MMS and incubated at 30°C for 2–3 days. (6b) rai1 mutants are not sensitive to HO endonuclease. WT (KRY622), rtt103 (KRY646), rai1 (KRY624), were induced with galactose for HO endonuclease to produce DSBs. The percentage survival on galactose compared to WT was calculated from three independent experiments and error bars denote SD.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
3.
Figure 2

Figure 2. RTT103 partially suppresses the the MMS sensitivity of yku70.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

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.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
4.
Figure 5

Figure 5. rtt103Δ show normal Rad53 phosphorylation.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

WT, yku70, rtt103, mec1 and rtt103mec1 null strains containing RAD53-Myc was treated with MMS for 2 h and anti-Myc western blots were performed. The slower moving fuzzy band indicates phosphorylation of Rad53. Fuzzy bands are visible in all lanes treated with MMS and contain wild type MEC1. mec1 mutants (lanes 9, 11) do not produce phosphorylated Rad53 upon MMS treatment. Wild type sample was loaded in two lanes of the gel for better comparison of the position of the fuzzy band. Same blot was probed with Sir2 antibody to show that separation of proteins was normal and identical in all lanes.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
5.
Figure 1

Figure 1. RTT103 partially suppresses the yku70 ts phenotype.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

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.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
6.
Figure 3

Figure 3. rtt103Δ are MMS sensitive and enhance the temperature sensitivity and MMS sensitivity of yku70 mutation.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

5 ul of ten-fold serial dilutions of wild type (KRY193), yku70 (KRY172), rtt103 (KRY285) and yku70rtt103 (KRY286) double mutants were spotted on YPD plates and incubated at 30°C, 35°C and 37°C (3a) or on plates containing MMS (3b) for 2 to 3 days. Two independent cultures for each mutant are shown here. rtt103Δ are sensitive to MMS and enhance both the temperature sensitivity and MMS sensitivity of yku70Δ. (3c) Quantification of MMS sensitivity. The MMS sensitivity of yku70, rtt103, rtt103 yku70 was quantified as described in Figure 2. The sensitivity to MMS for yku70, rtt103 and rtt103 yku70 were approximately 55, 100 and 750 fold respectively. Values plotted are from three independent cultures and error bars show SD.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
7.
Figure 8

Figure 8. Binding of Rtt103p to the site of DNA damage.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

(8a) Schematic diagram of the region around URA3 with two flanking I-SceI sites. Bars (1–12) represent the regions up to 3 kb away from I-SceI sites, at which Rtt103p binding was tested (8b) ChIP experiment to show Rtt103p binding at the site of damage. Rtt103Myc strain (KRY 448) was grown in galactose medium for three hours and then cross-linked with formaldehyde and followed by immunoprecipitation with anti-myc antibody. X-axis indicates the loci tested (see schematic) and y-axis shows the fold change of Rtt103p binding compared to SPS2 internal control; blue bars represent Rtt103p association just prior to galactose induction (no cut) and red bars represent association 3 hours after induction. SPS2 (13) and a region 10 kb from the telomere VI R (14) are negative controls. PMA1 3′ region (15) and ADH1 3′ region (16) where Rtt103 is reported to crosslink heavily are used as positive controls. Error bars denote SD of three independent immunoprecipitation experiments.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.
8.
Figure 4

Figure 4. rtt103Δ are sensitive to various kinds of DNA damage.. From: Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response.

(4a) rtt103Δ are sensitive to Sce-I endonuclease. Strains of WT (KRY304), yku70 (KRY376), rtt103 (KRY375) and rtt103 yku70 (KRY379) with two Sce-I sites on either side of the URA3 gene were induced with galactose to produce DSBs. The relative survival on galactose versus glucose was calculated from three independent cultures for each strain and error bars show SD. (4b) WT (KRY105), yku70 (KRY171), rtt103 (KRY230) and rtt103yku70 (KRY290) strains were transformed with supercoiled or linearized pRS313. The transformants were plated on SC-HIS plates in duplicates and incubated at 30°C for 2–3 days. The value plotted is the percentage of linear plasmid recovered relative to supercoiled plasmid for each strain from three independent transformation experiments.. (4c) Wild type (KRY105), yku70 (KRY171), rtt103 (KRY230) and rad1 (KRY473) strains were grown to mid log phase, 10-fold serially diluted and spotted on YPD plates. They were then exposed to UV radiation and incubated at 30°C for 2 days. (4d) rtt103Δ homozygous strains are severely defective in sporulation (i) and (ii) WT and rtt103 homozygous diploids were incubated on YPK plates for induction of sporulation and stained with DAPI to visualize nuclei after 4 days. rtt103Δ were transformed with either empty vector (iii) or with single copy of RTT103 (iv) and incubated on YPK plate for 4 days. DAPI images from this stage are shown. Quantification of spores was done by scoring 500 to 4000 cells.

Indukuri Srividya, et al. PLoS One. 2012;7(2):e31288.

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