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First published online 14 November 2007
doi: 10.1242/jcs.018366

Journal of Cell Science 120, 4209-4220 (2007)
Published by The Company of Biologists 2007
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The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast

Karine Dubrana*, Haico van Attikum{ddagger}, Florence Hediger§ and Susan M. Gasser

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland


Figure 1
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  		Fig. 1. Mec1 recruitment to the HO-endonuclease-induced DSB leads to the formation of large foci in most cells. (A) Crucial features of the yeast test strain JKM179 are shown. This strain lacks HM loci on chromosome 3 and contains an integrated galactose-inducible HO endonuclease gene. PCR fragments used to analyse ChIP experiments are labelled HO1, HO2 and HO3. (Graph) The efficiency of cleavage was quantified by PCR across the HO cleavage site, normalised to t=0 and plotted against time (hours after addition of 2% galactose). (B) The JKM179 strain was modified by an N-terminal Myc-epitope fusion to the genomic MEC1 gene, producing GA1529. Cells were prepared for IF after 2 hours of growth on glucose (0-hour time point) or after 0.5, 1, 2 or 4 hours on galactose. IF with anti-Myc (9E10 Mab, green) and anti-nuclear-pore (Mab414, red) was imaged on a Zeiss LSM510 confocal microscope. Single equatorial focal sections are shown. Only 70% of the nuclear volume is imaged and the percentage of such sections with a single bright Mec1 focus is indicated. In the lower right-hand panel, Myc-Mec1 (red) is visualised at 0 (inset) and 4 hours on galactose in a strain that carries a lacO array inserted 4.4 kb from the HO cut site and a GFP-lacI fusion (green). (C) ChIP for Myc-Mec1 at the indicated time points after induction of GAL::HO endonuclease on galactose. The 0-hour time point represents cells exposed to glucose for 2 hours to repress HO expression. Anti-Myc (9E10) and anti-HA (12CA5) were used for ChIP. DNA purified from input, the Myc-Mec1 (Myc) or HA (HA) IPs were analysed by multiplex PCR primers for the three HO sites shown in A and for the uncleaved SMC2 gene (see supplementary material Fig. S1). Quantitation of the products is presented as the ratio of the HO1, HO2 or HO3 signals to SMC2 in the IP, normalised to the same ratio in the corresponding input. In this way, changes in relative abundance of primer sites due to end-resection are factored out of the enrichment value. (D) Myc-Mec1 localisation as described in B in a derivative of JKM179 that lacks yKu70 (GA-1796). (E) Myc-Mec1 binding was analysed by ChIP as described in D using JKM179 (wild type, WT) and a derivative deleted for the gene encoding yKu70 (GA-1796). (F) Myc-Mec1 and yKu80-Myc binding was analysed by ChIP as described in C, but after 30 minutes on galactose in the JKM179 strain (WT, black and dark-grey bars) and in a derivative deleted for yku70 (light-grey bars). Bars, 1 µm.

 

Figure 2
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  		Fig. 2. Mec1 focus formation requires Ddc2, but not Mec1 or Rad53, kinase activity. (A) Myc-Mec1 localisation after HO induction for 0 and 4 hours in JKM179 derivatives deleted for both ddc2 and sml1 (GA1825) or for sml1 alone (GA1817). IF was performed with anti-Myc (9E10; green) and with affinity-purified anti-Sir4 (red) antibodies. Values correspond to the percentage of nuclei exhibiting a single bright Mec1 focus above a low background of diffuse Mec1. (B) Myc-Mec1 ChIP assay was performed as described in Fig. 1D using JKM179 derivatives carrying deletions for ddc2 sml1 or sml1 alone. (C) Myc-Mec1 localisation in JKM179-derived strains carrying deletions for sml1, for sml1 and rad53 together, or for sml1 with Myc-mec1-kd1 or Myc-mec1-kd2 mutations. IF for Myc-Mec1 (green, 9E10) and anti-pore (red, Mab414) is shown and the percentage of nuclei with one bright Mec1 focus is given. To the right, a JKM179 derivative expressing Myc-Rad53 was analysed by anti-Myc IF (green) after cut induction. Bars, 1 µm.

 

Figure 3
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  		Fig. 3. Ddc2-YFP foci disappear rapidly upon repair when donor loci are present for HR. (A) Ddc2-YFP fusion allows visualisation of the same Mec1-Ddc2 foci detected by IF after DSB induction (see Figs 1 and 2). We monitored the presence of Ddc2-YFP foci in two isogenic strains either bearing wild-type (WT) HML and HMR donor loci (KD-131; light-grey bars) or bearing hml and hmr deletions (GA-2358; dark-grey bars) after induction of the HO endonuclease on galactose for the indicated times. (B) Focus disappearance requires donor loci and Rad51. To show whether loss of foci coincides with repair by HR, we scored for Ddc2-YFP foci both in the strains used in panel A and in KD-132, in which wild-type HML and HMR are combined with a full rad51 deletion. Cells were grown on YPLGg overnight and GAL::HO was induced by 2% galactose for 1 hour. Thereafter, glucose was added to repress the endonuclease. The fluorescence image (bottom) shows a panel of typical cells bearing Ddc2-YFP (yellow) on a background of Nup49-CFP signal (red) after 1 hour of growth on galactose with 2 hours of glucose recovery. The corresponding phase image is also shown (top). (Graph) The percentage of cells with Ddc2-YFP foci were monitored in all strains after 1 hour of growth on galactose (gal), and then again after 2, 4 and 6 hours on glucose (glu). Note that the processing of DSBs continues even after the switch to glucose. Bar, 2 µm.

 

Figure 4
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  		Fig. 4. ssDNA is enriched in Myc-Mec1 precipitates. (A) Schematic for QAOS (Booth et al., 2001Go) near the HO cleavage site of MATalpha is shown. (B, left) The percentage of HO1 signal that exists as ssDNA was measured by QAOS in the input DNA at the indicated time points after HO cut induction. (Right) Myc-Mec1 ChIP was performed in JKM179 after galactose induction of the HO endonuclease. Fold enrichment of Mec1 over SMC2 at the HO2 site (0.6 kb away from the HO cleavage site) is shown. (C) The efficiency of HO cut-site cleavage (Fig. 1A, shown here in blue) is plotted against the percentage of ssDNA at HO1 in input DNA (Fig. 2B, red), the percentage of cells showing a single bright Mec1 focus (Fig. 1B, green) and the fold enrichment of Myc-Mec1 at the DSB over SMC2 (Fig. 1C, HO2, black). (D) Myc-Mec1 ChIP was performed after galactose induction of the HO endonuclease as in Fig. 1C. The IP DNA was then subjected to QAOS to quantify the fraction of the precipitated HO1 site that is ssDNA. The amount of ssDNA (light grey) is plotted as a fraction of the total Myc-Mec1-bound DNA. (E) Schematic of the degradation of Myc-Mec1-bound DNA by the RecJ exonuclease. RecJ is a 5'-to-3' exonuclease that degrades only ssDNA. It will not degrade DNA that is ds at the 5' end. (F) The amount of ssDNA at HO1 was measured by QAOS on Myc-Mec1-bound DNA at the indicated time points after HO induction either before or after incubation with RecJ endonuclease for 3 hours at 37°C, but after removal of proteins by proteolysis and phenol extraction.

 

Figure 5
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  		Fig. 5. Myc-Mec1 focus formation and recruitment in rad24, rfa1-t11 and rad51 mutants. (A) Myc-Mec1 localisation was determined by IF in JKM179 derivatives expressing Myc-tagged Mec1 (GA1529, shown in the insets in the rad9 panels), or in strains deleted for rad9, for rad24 or both, after 0 and 4 hours of HO induction on galactose. IF was conducted as in Fig. 1B. Values correspond to the percentage of nuclei exhibiting a single Mec1 focus. (B) Myc-Mec1 ChIP at the HO cleavage site as described in Fig. 1C using the rad9 rad24 double-deletion strain or the wild-type (WT) JKM179 derivative. (C) The percentage of ssDNA at the HO1 site was measured, after HO induction, by QAOS on total DNA from the JKM179-derivative GA1529 (WT), or from JKM179 with null alleles of rad51, rad9 rad24 or rfa1-t11. (D) IF as in Fig. 1B on the wild-type JKM179 derivative (GA1529) and isogenic strains bearing rfa1-t11 (GA2158) or a rad51 deletion (GA2163). Values correspond to the percentage of nuclei exhibiting a single bright Mec1 focus above the background of diffuse Mec1. Note that absence of a focus does not necessarily mean that no Mec1-Ddc2 binds the DSB but rather that the level is below the threshold of detection by IF. (E) Myc-Mec1 ChIP as described in Fig. 1C on JKM179 carrying either the rfa1-t11 mutation or a rad51 deletion. Results are shown for the HO2 probe only, although all probes showed analogous results. Bar, 1 µm.

 

Figure 6
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  		Fig. 6. A functional checkpoint is modulated by rfa1-t11 and Rad51. (A) Rad53 kinase activation was analysed by an in-gel autophosphorylation assay (Pellicioli et al., 1999Go) at the indicated time points after HO induction. Strains are JKM179 (wild type, WT) or derivatives thereof. Equal loading was scored on the same blot with anti-tubulin (TAT-1). (B) The wild-type and rfa1-t11 mutant cells were probed for checkpoint induction by monitoring the shift of phospho-Rad53-Myc (*) by SDS-PAGE. (C) The percentage of mononucleated large budded cells (G2/M arrest) was scored after ethanol fixation and DAPI staining at the indicated time points after HO induction. JKM179 strains and derivatives were used and values represent the mean of three experiments with >300 cells per set.

 

Figure 7
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  		Fig. 7. Steps leading to Mec1-Ddc2 accumulation and checkpoint activation by a DSB. Shown is a summary of the events involved in Mec1-Ddc2 accumulation at a DSB in budding yeast. yKu and the MRX complex bind rapidly and recruit Tel1 (ATM). At this early stage, a low level of Mec1-Ddc2 can also be detected. The resection of the end by Rad24 and the 9-1-1 complex (Rad17, Mec3 and Ddc1) leads to the loading of RPA. The OB-fold domain 1 of RPA recruits Mec1-Ddc2 and this is antagonised by Rad51. Resection and Mec1-Ddc2 recruitment both appear to be aided by Rad24/9-1-1. Sufficient accumulation of Mec1-Ddc2 becomes visible as a `focus' and the downstream checkpoint activation of Rad53, stimulated by Rad9, occurs.

 

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© The Company of Biologists Ltd 2007