Yeast experiments

Yeast strains

Yeast strains used in this study are shown in table 2.3. RSC2 was deleted in the DMY3010, W1479-11C and W1490-16A strains via standard gene-disruption methods using a cassette amplified by PCR.

Analysis of Rsc1 and Rsc2 complex composition (Anna Chambers)

Rsc1-TAP and Rsc2-TAP were purified by two-step affinity purification. 1 litre of yeast was grown to mid-late log phase, pelleted and popcorn made in liquid nitrogen. Yeast were lysed by grinding and the powder was resuspended in 4 pellet volumes of IgG binding buffer (10 mM Tris-HCl pH8, 300 mM NaCl, 0.1% NP40, 100 µg/ml PMSF, 1 µM leupeptin, 1 µg/ml pepstatin A, 0.5 µg/ml aprotinin). Lysate was centrifuged at 15,000 rpm for 10 min at 4°C before the supernatant was incubated with 200 ul of IgG Sepharose beads for 2 hours at 4°C. Beads were washed with 3 X with 1 ml IgG binding buffer, followed by 2 X 1 ml TEV cleavage buffer (10 mM Tris-HCl pH8, 150 mM NaCl, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT100 µg/ml PMSF, 1 µM leupeptin, 1 µg/ml pepstatin A, 0.5 µg/ml aprotinin). Beads were resuspended in 1 ml TEV cleavage buffer, 10 ul TEV protease were added to the samples, which were incubated overnight

Table 2.3. Yeast strains used in this study

Strain name Genotype Source

JKM179 Δho Δhml::ADE1 Δhmr::ADE1 ade1-100 leu2-3,112 Lee et al. 1998

trp1::hisG lys5 ura3-52 ade3::GAL::HO MATα

YNK179-177 ∆rsc1::KanMX in JKM179 Kent et al. 2007

YNK179-191 ∆rsc2::KanMX in JKM179 Kent et al. 2007

YPF17 ∆mata::hisG ade1 lys5 trp1::hisG ura3-52 leu2::HOcs ∆ho Paques et al. 1998

∆hml::ADE1 ∆hmr::ADE1 ade3::GAL::HO

YNK17-20 ∆rsc1::KanMX in YPF17 Chambers et al. 2012

YNK17-24 ∆rsc2::KanMX in YPF17 Chambers et al. 2012

DMY2804 RDN1-NTS1::mURA3 in W303a Huang et al. 2006

JDY789 ∆rsc1::KanMX in DMY2804 Chambers et al. 2012

JDY790 ∆rsc2::KanMX in DMY2804 Chambers et al. 2012

DMY3010 RAD5 RDN1::ADE2 in W303a Huang et al. 2006

JDY964 ∆rsc2::KanMX in DMY3010 Brownlee et al. 2014

W1479-11C MAT::HIS3 leu2Δ::EcoRI::URA3-HOcs::leu2ΔBstEII Smith & Rothstein 1999

∆rsc2::KanMX in W1479-11C This study

W1490-16A MAT::HIS3 RAD5 leu2Δ::BstEII::URA3-HOcs::leu2ΔEcoRI Smith & Rothstein 1999

at 4°C. 3 ml CaM binding buffer (10 mM Tris-HCl pH8, 150 mM NaCl, 10 mM beta- mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% NP40, 100 µg/ml PMSF, 1 µM leupeptin, 1 µg/ml pepstatin A, 0.5 µg/ml aprotinin) and 3 µl 1 M CaCl2 were added, and beads collected by centrifugation. The supernatant was bound to 50 µl calmodulin beads for 1 hour at 4°C, washed with 4 X 1 ml CaM wash buffer (CaM binding buffer with 300 mM rather than 150 mM NaCl), and eluted in 1 ml elution buffer (10 mM Tris-HCl pH8, 150 mM NaCl, 10 mM beta-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% NP40, 30 mM EGTA, 100 µg/ml PMSF, 1 µM leupeptin, 1 µg/ml pepstatin A, 0.5 µg/ml aprotinin). 20% ice- cold acetone was used to precipitate proteins and the pellet was resuspended in 20 µl 10 mM Tris-HCl pH 7.5. A mock purification was performed from lysate created from an isogenic untagged strain. Gel slices were analyzed in the University of Sussex Proteomics Centre.

qPCR analysis of transcriptional induction (Anna Chambers)

For analysis of RNR induction, cultures of JKM179, rsc1, rsc2 and mec1 mutant strains were grown to mid-log in YPAD. Cultures were then incubated for a further 3 hours at 30°C in the absence or presence of 0.05% MMS. RNA was reverse transcribed using Qiagen QuantiTech RT kit. For each sample, qPCR reactions were performed using primers within RNR2, RNR3 and results were normalized to the ACT1 locus. The transcript level of the normalized wild type undamaged sample is set to 1 and all other values are shown relative to this. For analysis of HXT7 transcription, cultures of rsc2 DMY3010 containing cancer mutation plasmids were grown to mid-log phase in SC lacking tryptophan and RNA extracted using an RNeasy kit (Qiagen). 1µg of RNA was reverse transcribed into cDNA for analysis by qPCR using primers specific to the HXT7 locus.

G2/M checkpoint analysis by fluorescence-activated cell sorting (FACS) (Anna Chambers)

Cultures of wild-type, rsc1 and rsc2 JKM179 cells were grown to mid-log in YPAD and an asynchronous sample taken at the outset of the assay. Cells were arrested in G2/M by incubation with 15mg/ml nocodazole for 1hr 45 minutes. Cultures were incubated for a further 45 minutes (with nocodazole still present) in the presence or absence of 0.1% MMS. Yeast were washed and resuspended in fresh YPAD and fixed in ethanol at 0, 30, 60, 90, 120, 150, 180 and 210 minutes after release. Cells were harvested and

incubated in 1mg/ml RNase A for 4 hours at 37°C, followed by incubation for 1 hour at 50°C in 2mg/ml proteinase K solution. Yeast cells were collected by centrifugation and resuspended in FACS buffer (200mM Tris-HCl pH7.5, 200mM NaCl, 78mM MgCl2).

Cells were added to 50µg/ml propidium iodide in 50mM Tris-HCl pH7.5, sonicated and analysed using a FACS Calibur.

G1 checkpoint analysis by fluorescence-activated cell sorting (FACS)

Cultures of wild-type, rsc1 and rsc2 DMY2804 cells were grown to mid-log in YPAD and an asynchronous sample taken at the outset of the assay. Cells were arrested in G1 by incubation with 4µg/ml α-factor for 1hr followed by an additional 2µg/ml for another hour. Cultures were incubated for a further 1hr with a final 2µg/ml dose of α- factor in the presence or absence of 0.1% MMS. Samples were taken at 0 and 30 minutes following addition of MMS and fixed in ethanol before washing and resuspending the remaining culture in fresh YPAD. Subsequent samples were taken at 60, 90, 120, 150, 180, 210, 240 and 270 minutes. Cells were harvested and incubated in 1mg/ml RNase A for 4 hours at 37°C, followed by incubation for 1 hour at 50°C in 2mg/ml proteinase K solution. Yeast cells were collected by centrifugation and resuspended in FACS buffer (200mM Tris-HCl pH7.5, 200mM NaCl, 78mM MgCl2).

Cells were added to 50µg/ml propidium iodide in 50mM Tris-HCl pH7.5, sonicated and analysed using a FACS Calibur.

Genomic NHEJ assays

Mid-log phase cultures of JKM179 or YPF17 were grown in YPAD or, when plasmids were used, SC lacking leucine, serially diluted and plated onto YPAD or SC lacking leucine containing both 2% glucose and 2% galactose. Colonies were counted after 4 days incubation at 30°C and repair efficiency was calculated as survival on galactose relative to survival on glucose. Experiments were performed at least in triplicate. Repair junctions of colonies surviving on galactose were analysed by sequencing.

Spot tests

Mid-log phase cultures of wild-type, rsc1 and rsc2 JKM179 containing plasmids were grown in SC lacking leucine, and cultures of rsc2 DMY3010 containing plasmids were grown in SC lacking tryptophan. Cultures were diluted to OD600=0.2 and 5-fold serial

were grown for 2 days at 30°C and spot tests were performed multiple independent times.

Cell cycle survival assays

Exponentially growing cultures of wild-type, rsc1, rsc2 and rad52 JKM179 in YPAD were arrested in nocodazole, treated with MMS, centrifuged and resuspended in fresh YPAD as for G2 checkpoint analysis by FACS. Serial dilutions were plated onto YPAD and colonies were counted 3 days after incubation at 30°C. Experiments were performed in duplicate by Anna Chambers. For G1 survival assay, exponentially growing cultures of wild-type, rsc1 and rsc2 DMY2804 in YPAD were arrested in α- factor, treated with MMS, centrifuged and resuspended in fresh YPAD as for G1 checkpoint analysis by FACS. Serial dilutions were plated onto YPAD and colonies were counted 3 days after incubation at 30°C. Experiments were performed in triplicate.

Indirect end-label analysis (Sam Durley and Tracey Beacham)

Chromatin digestion using micrococcal nuclease (MNase) was performed exactly as described by Kent et al. 2007. Yeast cultures were grown in 1% peptone, 1% yeast extract plus 2% D(+)-raffinose. Cells were grown at 30 °C to densities of ∼2.0 × 107

nucleated cells/ml (determined by hemocytometry). Cells were sampled just before (“HO 0 min” samples) and at time points after HO induction by the addition of D(+)- galactose to 2%. Spheroplasts were created by digestion in 1 ml of 10 mg/ml Arthrobacter luteus yeast lytic enzyme in 1M sorbitol, 5 mM β-mercaptoethanol for 4

min at 22 °C. Samples containing 2.0 × 108 _{nucleated cells were digested with between}

75 and 300 units/ml MNase at 37 °C for 3 min or processed for purification of deproteinized DNA. Deproteinized DNA samples were digested with 5 units/ml MNase at 22 °C for 10 s to create “DNA” control digests. Purified DNA samples were digested

to completion with BglII or BspEI (for analysis of the LEU2 and MAT loci, respectively)

and separated on 1.5% agarose gels. Southern blots of the gels were hybridized to 400-bp indirect end label probes, which abut the relevant restriction sites.

Yeast survival and rDNA recombination assays

Wild-type and rsc2 DMY3010 strains were grown to mid-log in YPAD before diluting to OD600=0.2 and serial dilutions plated onto SC plates containing 10µg/ml adenine. rsc2

DMY3010 cells containing plasmids were grown in SC lacking tryptophan and plated onto SC plates lacking tryptophan containing 10µg/ml adenine. Plates were incubated at 30°C for 3-4 days before being placed at 4°C to intensify the red colouration. The percentage rate of ADE marker loss was calculated by dividing the number of half red/white sectored colonies with the total number of colonies per plate, excluding red colonies. Counts for each experiment were from six individual colonies per strain. Standard deviations are given ± the mean from three separate experiments. Between 10,000 and 20,000 colonies were counted for each strain in total.

LEU2 direct-repeat recombination assay

Wild-type and rsc2 colonies from W1479-11A and W1490-16A strains grown on YPAD were resuspended in water and plated onto SC plates and SC plates lacking leucine. Colonies were counted after incubation at 30°C for 3-4 days. The rate of LEU+ prototroph formation in each experiment was calculated using the method of the median (Lea & Coulston 1949) from eight individual colonies. Standard deviations are given ± the mean from three separate experiments.

Yeast growth curves (Anna Chambers)

Cultures of rsc2 DMY3010 containing cancer mutation plasmids were grown to mid-log phase in SC lacking tryptophan and diluted to OD600=0.05 into fresh media. The OD600

was measured every hour for 9 hrs.