Yeast Transcription Termination Factor Rtt103 Functions in DNA Damage Response

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

Binding of Rtt103p to the site of DNA damage.

<p>(<b>8a</b>) Schematic diagram of the region around <i>URA3</i> 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 (<b>8b</b>) 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 <i>SPS2</i> internal control; blue bars represent Rtt103p association just prior to galactose induction (no cut) and red bars represent association 3 hours after induction. <i>SPS2</i> (13) and a region 10 kb from the telomere VI R (14) are negative controls. <i>PMA1</i> 3′ region (15) and <i>ADH1</i> 3′ region (16) where Rtt103 is reported to crosslink heavily are used as positive controls. Error bars denote SD of three independent immunoprecipitation experiments.</p

<i>rtt103</i>Δ show normal Rad53 phosphorylation.

<p>WT, <i>yku70</i>, <i>rtt103</i>, <i>mec1</i> and <i>rtt103mec1</i> null strains containing <i>RAD53-Myc</i> 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. <i>mec1</i> 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.</p

<i>rtt103</i>Δ do not show hyper-recombination phenotype.

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

<i>RTT103</i> partially suppresses the the MMS sensitivity of <i>yku70.</i>

<p>WT (KRY193) and <i>yku70</i> (KRY 172) mutants were transformed with empty vector, KM93 and <i>RTT103.</i> 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. (<b>2b</b>) Quantification of MMS sensitivity. The MMS sensitivity of <i>yku70</i> was quantified by plating appropriate dilutions on plates with and without MMS. Sensitivity of WT was set to 1. <i>yku70</i> is approximately 60 fold sensitive and upon overexpression of <i>RTT103</i> the sensitivity to MMS is reduced by approximately 8 fold. Quantification was done for three independent cultures and error bars show SD.</p

Figure 6

<p>(<b>6a</b>) <i>rat1-1</i> and <i>rai1</i> mutants are not sensitive to MMS. WT (KRY105), <i>rtt103</i> (KRY230) WT <i>RAI1</i> (KRY631), <i>rai1</i> (KRY632), WT <i>RAT1</i> (KRY633) and <i>rat1-1</i> (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. (<b>6b</b>) <i>rai1</i> mutants are not sensitive to HO endonuclease. WT (KRY622), <i>rtt103</i> (KRY646), <i>rai1</i> (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.</p

<i>rtt103</i>Δ are sensitive to various kinds of DNA damage.

<p>(<b>4a</b>) <i>rtt103</i>Δ are sensitive to <i>Sce</i>-I endonuclease. Strains of WT (KRY304), <i>yku70</i> (KRY376), <i>rtt103</i> (KRY375) and <i>rtt103 yku70</i> (KRY379) with two <i>Sce</i>-I sites on either side of the <i>URA3</i> 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. (<b>4b</b>) WT (KRY105), <i>yku70</i> (KRY171), <i>rtt103</i> (KRY230) and <i>rtt103yku70</i> (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.. (<b>4c</b>) Wild type (KRY105), <i>yku70</i> (KRY171), <i>rtt103</i> (KRY230) and <i>rad1</i> (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. (<b>4d</b>) <i>rtt103</i>Δ homozygous strains are severely defective in sporulation (<b>i</b>) and (<b>ii</b>) WT and <i>rtt103</i> homozygous diploids were incubated on YPK plates for induction of sporulation and stained with DAPI to visualize nuclei after 4 days. <i>rtt103</i>Δ were transformed with either empty vector (<b>iii</b>) or with single copy of <i>RTT103</i> (<b>iv</b>) 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.</p

List of the yeast strains used in this study.

<p>List of the yeast strains used in this study.</p

<i>RTT103</i> partially suppresses the <i>yku70</i> ts phenotype.

<p>WT (KRY193) and <i>yku70</i> (KRY172) mutants were transformed with empty vector, KM93 and <i>RTT103.</i> 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. (<b>1b</b>) Quantification of temperature sensitivity. The temperature sensitivity of <i>yku70</i> was quantified by plating out appropriate dilutions of 3 independent cultures at the appropriate temperatures. Sensitivity of WT was set to 1. <i>yku70</i> is approximately 40 fold sensitive and upon overexpression of <i>RTT103</i>, the sensitivity to temperature is reduced by approximately 7 to 8 fold. Error bars indicate SD.</p

<i>rtt103</i>Δ are MMS sensitive and enhance the temperature sensitivity and MMS sensitivity of <i>yku70</i> mutation.

<p>5 ul of ten-fold serial dilutions of wild type (KRY193), <i>yku70</i> (KRY172), <i>rtt103</i> (KRY285) and <i>yku70rtt103</i> (KRY286) double mutants were spotted on YPD plates and incubated at 30°C, 35°C and 37°C (<b>3a</b>) or on plates containing MMS (<b>3b</b>) for 2 to 3 day<b>s</b>. Two independent cultures for each mutant are shown here. <i>rtt103</i>Δ are sensitive to MMS and enhance both the temperature sensitivity and MMS sensitivity of <i>yku70</i>Δ. (<b>3c</b>) Quantification of MMS sensitivity. The MMS sensitivity of <i>yku70</i>, <i>rtt103</i>, <i>rtt103 yku70</i> was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031288#pone-0031288-g002" target="_blank">Figure 2</a>. The sensitivity to MMS for <i>yku70</i>, <i>rtt103</i> and <i>rtt103 yku70</i> were approximately 55, 100 and 750 fold respectively. Values plotted are from three independent cultures and error bars show SD.</p