Rad10 protein in the yeast S. cerevisiae
was tagged at its C-terminal end by integrating the gene encoding the yellow-shifted version of green fluorescent protein from Aequorea victoria
in-frame at the RAD10
chromosomal locus, using adaptamer-mediated PCR as previously described (15
). The resulting yeast strain expresses a Rad10-YFP fusion protein (Rad10-YFP) that migrates as a 54 kDa polypeptide during SDS PAGE, whereas the untagged protein migrates as a 24 kDa band (compare lanes 2 and 4 with lanes 5–7 in A). A control lane containing GFP protein alone (lane 1) exhibits no signal with α-Rad10 antibody, and extracts from a rad10
deletion mutation (rad10Δ
, lane 3) exhibit only background signal of the antibody. As expected, by fluorescence microscopy, the Rad10-YFP strain exhibits yellow fluorescence in the nucleus as indicated by colocalization of the YFP signal with DAPI-stained DNA in the nucleus (B).
Figure 1. (A) The Rad10-YFP strain exhibits an increased polypeptide size consistent with Rad10 expressed as a YFP fusion protein. Immunoblot of WCE from the indicated yeast strains probed with a Rad10 antibody. 1 µg total protein was loaded to each lane. (more ...)
Next, the functionality of the Rad10-YFP protein in vivo was examined. A plasmid-based assay was used to test Rad10-YFP function in SSA and GC (A). Densitometric analysis of genome blot results (B) shows that the efficiencies of SSA and GC are somewhat lower (86 and 54%, respectively) in the YFP-tagged strain compared with wild-type. However, the fluorescent label does not compromise the function of Rad10 protein during NER, as evidenced by examining the UV radiation sensitivity of the Rad10-YFP strain. Indeed, the Rad10-YFP strain exhibits wild-type levels of survival following exposure to UV-C irradiation (C).
Figure 2. (A) Schematic showing the SSA assay reporter plasmid [modified from (17)]. The reporter plasmid, pJF6 can be repaired by either SSA or GC. Triple digestion of the plasmid by PstI, SmaI and HindIII gives 5.6 and 4.4 kb bands, respectively, that hybridize (more ...)
To determine whether Rad10 protein is recruited to DSBs, such lesions were generated in vivo
in a strain containing both Rad10-YFP and a unique DSB labeling system previously reported (A) (4
). Specifically, a yeast strain was prepared that expresses the coding sequence for the TetR fused to monomeric red fluorescent protein (TetR-RFP). The strain additionally contains a DNA cassette with 224 copies of the TetR binding sequence abutted with one copy of the I-Sce
I restriction endonuclease site. I-Sce
I enzyme was introduced into the strain via a selectable extrachromosomal plasmid under the control of the GAL1
promoter (pWJ1320) (4
). Subsequent induction of I-Sce
I generates a single DSB in close physical proximity to many copies of TetR-RFP protein bound to DNA, thereby enabling visualization of the DSB site as a bright red focus (DSB-RFP) within the nucleus (B). Such induction of DSBs results in increased punctate yellow foci (B, Rad10-YFP panel), indicating the presence of a high local concentration of Rad10-YFP. A significant subset of Rad10-YFP foci colocalized with DSB-RFP foci in DSB-induced cells, indicating that Rad10-YFP protein is indeed recruited to DSB sites (B and ). Rad10-YFP foci were also observed spontaneously in uninduced cells, but in fewer numbers and they rarely colocalized with the DSB site (see below). Some of these spontaneous events likely reflect the recruitment of the tagged protein to spontaneous DSBs, as previously reported for proteins of the RAD52
epistasis group, suggesting that Rad10 is recruited to both endonuclease-induced and naturally occurring DSBs (4
). Indeed, cells co-expressing Rad10-YFP and Rad52-CFP exhibited spontaneous Rad52-CFP foci that colocalized with Rad10-YFP (). Since spontaneous Rad10 foci were consistently observed at a higher frequency (70–80%) than spontaneous Rad52 foci (5–10%), many of the Rad10 foci likely reflect the involvement of Rad10 in other DNA repair pathway(s), such as NER and MMEJ.
Figure 3. (A) Scheme showing experimental design. The I-SceI gene is introduced to the cells on an adenine selectable (ADE) plasmid under the control of the galactose-inducible (GAL1) promoter. Upon induction with galactose, the I-SceI gene is switched on to produce (more ...)
Figure 4. Rad10-YFP foci are induced in response to DNA DSBs and colocalize with the DSBs. Yeast strains (PF025-7A, WPF021-4A, PF030-49A and PF023-15A) containing the Rad10-YFP/DSB-RFP labeling system were transformed with plasmid pWJ1320, and cultures of transformants (more ...)
To determine the cell cycle dependence of Rad10 foci, cells were scored not only for Rad10-YFP and DSB-RFP foci, but also for cell cycle phase (S, G2/M or G1, see ‘Materials and Methods’ section). S phase cells manifested the greatest magnitude of DSB-colocalized Rad10 foci, exhibiting approximately a 12-fold increase in DSB-induced cells over that in uninduced controls (, wild-type). Colocalized foci were also induced in G2/M and G1 cells, but to a much lesser extent (), suggesting that the repair of DSBs by HR transpires primarily during S phase of the cell cycle, presumably due to ongoing functions of Rad10 such as repair of spontaneous DSBs and NER. The great majority (70–80%) of uninduced cells contained at least one Rad10-YFP focus. However, upon DSB induction the increase in the numbers of Rad10-YFP foci matched the increase in the numbers of Rad10-YFP foci colocalized to DSB sites.
We next examined the genetic requirements of recruitment of Rad10-YFP to DSBs. Isogenic strains deleted for the MRE11, RAD52 or RAD51 genes (mre11Δ, rad52Δ or rad51Δ) were transformed with the I-SceI expression plasmid, pWJ1320. DSBs were induced and the formation of DSB-colocalized Rad10-YFP foci was monitored in parallel with wild-type controls. The frequency of DSB-colocalized Rad10-YFP foci observed in the mre11Δ strain was not appreciably altered relative to the wild-type, (P-values > 0.19 in all phases of the cell cycle) indicating that Mre11 protein is not required to recruit Rad10-YFP to DSBs (, compare black wild-type and mre11Δ bars). Hence, while visual examination of suggests a small reduction in the number of Rad10-YFP foci colocalized with DSB-RFP foci in the mre11Δ background, this difference is not statistically significant under the experimental conditions employed. In contrast, rad52 and rad51 deletion mutants exhibited very few Rad10-YFP foci localized to sites of DSBs upon I-SceI induction (, compare wild-type, rad52Δ and rad51Δ bars).
To determine whether Rad10 colocalizes with Rad52 or Rad51 at DSB sites, DSB induction experiments were conducted using triple-labeled Rad10-YFP/DSB-RFP/Rad52-CFP or Rad10-YFP/DSB-RFP/Rad51-CFP strains (A). Triple-labeled experiments were imaged as single focal planes owing to photobleaching of the Rad10-YFP chromophore which precluded the formation of Z-stack images (see ‘Materials and Methods’ section). Accordingly, the overall percentage of cells containing Rad10-YFP is reduced in single-plane images (B) versus Z-stack images (), since a single focal plane reveals only a portion of the total thickness of the average cell.
Figure 5. (A) A strain containing Rad10-YFP, the DSB-RFP labeling system and Rad52-CFP (WPF019-26C) was transformed with plasmid pWJ1320 and cultures of transformants induced with galactose as described in ‘Materials and Methods’ section. Single-focal (more ...)
In both strains, colocalization of all three labels (YFP, RFP and CFP) was observed, indicating that Rad10-YFP is present at DSB sites at the same time as Rad52-CFP (A) or Rad51-CFP (data not shown). Furthermore, ~75% of the Rad10-YFP foci that colocalized with the DSB-RFP marker were also colocalized with Rad52-CFP, both in S and G2/M phase cells, while essentially no G1 cells exhibit similar triple colocalization (B). Hence, most of the Rad10-YFP foci localized to the induced DSBs also had Rad52-CFP present indicating considerable temporal overlap of the functions of Rad10 with Rad52. Altogether, we conclude that Rad10 protein colocalizes with Rad51 and Rad52 proteins at DSBs during S and G2/M phases of the cell cycle.
Since the repair of DSBs induced by a restriction enzyme can differ from the repair of breaks induced by IR (20
), a series of experiments were done using a gamma source to induce DSBs. Experiments were conducted in both RAD51
strains containing Rad10-YFP and Rad52-CFP. Two hours post-irradiation, individual and colocalized foci were counted and compared with results from non-irradiated controls. Following IR, the numbers of cells containing at least one Rad10-YFP focus increased similarly in both the RAD51
and the rad51Δ
strains relative to non-irradiated controls (1.3- and 1.7-fold, respectively; data not shown). The numbers of cells containing at least one Rad10-YFP/Rad52-CFP colocalized focus also increased in both strains relative to non-irradiated controls (6.2-fold for RAD51
and 4.2-fold for rad51Δ
, respectively; data not shown). As with the I-Sce
I experiments, most of the foci were observed in the S, G2 or M phases of cell cycle. Upon IR induction, the number of S, G2 or M phase cells containing at least one colocalized Rad10-YFP/Rad52-CFP focus increased 4.8-fold for RAD51
cells and 2.5-fold for rad51Δ
cells (). Thus, IR induction of colocalizing Rad10 and Rad52 foci is RAD51
-independent unlike that observed following I-Sce
I induction. Interestingly, in the absence of IR induction, the number of S, G2 and M phase cells containing at least one spontaneous colocalized Rad10-YFP/Rad52-CFP focus was 2.5-fold higher in rad51Δ
cells than in RAD51