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Monitoring S. pombe genome stress by visualizing end-binding protein Ku

Studies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.


Construction of strains with fluorescently tagged Pku70 and Mre11 55
Ku (a heterodimer of Pku70/80) and MRN (Mre11/Rad50/Nbs1) protein complexes are 56 known for high affinity for binding DNA ends (Fell, V. L., & Schild-Poulter, C. 2015;Shibata, 57 A., et al. 2018). We tagged Pku70 on its C-terminal end with Citrine fluorescent protein and 58 integrated into the endogenous locus (see methods). Using a similar strategy we also tagged 59 Mre11 on its C terminal end with mCherry fluorescent protein. The resulting strains were 60 compared to wild type, pku70∆ , mre11∆ and rad51∆ for their growth on four typical genotoxic 61 drugs: methyl methanesulfonate (MMS), which creates alkylation damage that inhibits DNA replication fork progression; camptothecin (CPT), which blocks Topoisomerase I cleavage; 63 hydroxyurea (HU), which causes nucleotide starvation and fork pausing; and Phleomycin 64 (phleo), a radio-mimetic that causes single-and double-strand breaks. Both the Mre11-mCherry 65 and Pku70-Citrine tagged strains behaved the same as WT under normal growth and genotoxic 66 stress. The Δpku70 strain also shows no sign of genotoxin sensitivity, as reported previously 67 (Manolis, K. G., et al. 2001;Sánchez, A., & Russell, P. 2015). (Fig. 1A) 68 69 Pku70 and Mre11 have increased nuclear signal following genotoxic stress 70 When we imaged the tagged strains under normal growth conditions, we observed a few 71 scattered foci in Pku70-citrine cells and diffuse nuclear fluorescence in Mre11-mCherry cells 72 ( Fig. 1B). We next examined the distribution of signal in cells treated with MMS, CPT,Phleo,73 or HU at 32ºC after 4 hours. There is a significant increase of cells with individual Pku70 nuclear 74 foci in MMS, CPT, and to a lesser extent Phleo. Cells treated with HU did not show any 75 significant difference from WT (Fig. 1C). In contrast, the Mre11-mCherry signal showed diffuse 76 pan-nuclear staining in untreated cells ( Fig 1B). Following 4 hours of treatment with the four 77 genotoxic drugs, Mre11-mCherry did not show obvious foci. Rather, we observed generalized 78 areas of increased fluorescence over threshold, but these typically were not well-defined discrete 79 puncta as seen with other markers. 80 81

Colocalization of Pku70 and Mre11 with other markers of DNA damage 82
Previous studies of genome instability in fission yeast have imaged the single stranded 83 binding protein Ssb1 (Rad11, RPA) and the homologous recombination protein Rad52 in 84 response to different forms of replication stress (Meister, P., et al. 2003;Kilkenny, M. L., et al 85 2008;Carneiro, T., et al. 2010;Bass, K. L., et al., 2012;Sabatinos, S. A., et al. 2012). We 86 examined co-localization using CPT, MMS, HU and Phleo in a strain with Pku70-citrine, Rad52-87 mCherry, and RPA-CFP. Four hours after drug addition at 32ºC, we determined frequency of 88 colocalization among all three tagged proteins. While there was partial overlap, Ku is not 89 completely concordant with the other markers ( Fig. 2A). 90 The number of foci per nucleus was calculated and binned as either 1or ≥ 2 foci using an 91 automatic foci counter in ImageJ as described in the materials and methods (Fig. 2B). We 92 observed that CPT 20µM contained the highest frequency of Pku70 foci, then MMS, Phleo, and 93 MMS was lowered from .9mM to .45mM to better resolve single foci. 96 Colocalization was determined using the objects-based method in the ImageJ pluggin 97 JACoP (see materials and methods). Fig. 2C shows the proportion of Pku70-Citrine foci that 98 overlap with a thresholded region for Rad52-mCherry or RPA-CFP. For CPT, MMS, and Phleo 99 these proportions vary from 60-90%. In contrast, the scattered foci in HU showed only about 100 30% of Ku co-associating with another marker. Fig 2D shows  We performed a similar study with Mre11-mCherry but could not perform the same 104 quantitation because Mre11-mCherry does not form discrete foci. We observed areas of 105 generally increased fluorescence but never clear puncta as with Pku70, Rad52, or RPA. 106 Observing these cells in three-dimensional reconstruction showed no obvious colocalization 107 between Rad52-YFP and RPA-CFP in live cell video microscopy, or in static images 108 (Supplemental Fig. 1; Fig. 3A,B). 109 110

Pku dynamics in S phase specific damage 111
The genotoxin MMS causes alkylation damage, generating lesions that block DNA 112 polymerase (Lundin, C., et al. 2005). This typically results in replication template switching 113 (Barbour and Xiao 2003;Andersen et al. 2008). Previous work has suggested that Ku is 114 recruited by blocked and regressed replication forks (Teixeira-Silva, A., et al. 2017). Therefore, 115 we investigated the dynamics of Ku response to MMS treatment as a model for disruptions in 116 fork progression. We used live cell video microscopy to observe cells containing Rad52-117 mCherry and Pku70-Citrine over a 5hr period of MMS treatment at 28ºC. We observe distinct 118 dynamics for Rad52 and Pku70 recruitment during treatment. While absolute timing differs in 119 individual cells, typically a Ku focus appears for a short time and partially co-localized with 120 Rad52. 121 Rad52 and single strand DNA binding protein Rad11, a subunit of RPA (Meister, P., et al., 2003;137 Carneiro, T., et al. 2010;Sabatinos, S. A., et al. 2012). Studies have shown that in cycling wild 138 type cells, approximately 10-20% of cells show evidence of single RPA or Rad52 foci, likely due 139 to sporadic S phase events. These proteins show distinct patterns in response to genotoxic 140 stresses induced by mutations in the replication or repair pathways (Sabatinos, S. A., et al. 2012;141 Sabatinos, S. A., et al. 2015;Ranatunga, N. S., & Forsburg, S. L. 2016), or in response to 142 exogenous agents such as hydroxyurea (HU), which causes replication fork stalling (Thelander, 143 L., & Reichard, P. 1979); MMS, an alkylating agent that generates lesions that block the 144 replication fork (Lundin, C., et al. 2005); camptothecin (CPT), a topoisomerase I inhibitor that 145 leads to S-phase specific double strand breaks (Li, T. K., & Liu, L. F. 2001);and bleo-or 146 phleomycin, radiomimetic drugs that causes single-and double-strand breaks (Povirk, L. F. 147 1996). 148 The current study seeks to expand the library of tagged proteins, part of our strategy to 149 develop a fingerprint for the response to different forms of genotoxic stress. We investigated 150 fluorescently tagged Mre11 and Pku70 as markers for DNA breaks. 151 The MRN complex is one of the earliest responders to DSBs (Shibata, A., et al. 2018;152 Syed, A., & Tainer, J. A. 2018). and is essential to drive resection (Wang, Q., et al. 2014;153 Shibata, A., et al. 2018;Langerak, P., et al. 2011;Teixeira-Silva, A., et al. 2017). Our Mre11-154 mCherry construct showed a diffuse pan-nuclear signal in untreated cells. We did not see obvious focus formation of Mre11-mCherry following treatment with genotoxins. Rather, it 156 maintained a diffuse signal with regions of brightness. In other systems, MRN has been shown to 157 be an immediate responder to double strand breaks induced by ionizing radiation (Maser, R. S., 158 et al. 1997). Our failure to see this form of localization may indicate the timing of our analysis, 159 and diffuse distribution of lesions in drug treated cells, compared to concentrated sites of damage 160 from of ionizing radiation. 161 Previous whole-cell localization of Pku70 in S. pombe was carried out using C terminal 162 epitope tagged Pku70 and immunofluorescence on fixed cells (Manolis, K. G., et al. 2001). In 163 unperturbed cells, there is a diffuse pan-nuclear localization. Association of Ku with DNA ends 164 has been investigated using ChIP methods; in wild type cells, it is not enriched unless the MRN 165 complex is missing (Langerak, P., et al. 2011;Teixeira-Silva, A., et al. 2017). Visualization of 166 Pku70 in live fission yeast cells has not previously been performed. 167 We saw few Ku foci in WT cells, consistent with previous reports. Treatment for 4 hours 168 with our panel of genotoxins showed that HU has little to no accumulation of Ku foci. Treatment 169 with CPT causes a modest but limited increase in the fraction of cells with foci at 10µM and a 170 much more drastic increase at 20µM. Similarly, phleomycin, a radiomimetic that causes DNA 171 breaks throughout the cell cycle, has a modest but limited increase in foci. 172 The most dramatic increase in fraction of cells with foci was observed with MMS at 173 0.9mM, an alkylating agent that results in error-free and error prone base excision repair during 174 S phase, and thus leading to trans lesion synthesis. This induction in MMS is consistent with 175 prior observations suggesting that Ku is recruited to regressed or broken replication forks in 176 order to stabilize the free end (Langerak, P., et al. 2011;Teixeira-Silva, A., et al. 2017). This 177 suggests that even in MRN + cells, there are situations where Ku remains associated with sites of 178 genome stress. 179 We observed a substantial colocalization between RPA or Rad52 and Ku, in cells treated 180 with MMS, CPT, or Phleo. This result was a surprise as many models suggest Pku should be 181 removed by the time resection and recombination proteins are recruited. One possibility for the S 182 phase specific toxins is that Pku could be binding to reversed forks at repair centers. Previous 183 studies suggest that Pku plays a role at reversed forks in order to maintain genome stability, 184 particularly in cells with defective HR repair such as brc1Δ (Sánchez, A., & Russell, P. 2015; exonuclease activity, including helicase unwinding and strand invasion. 187 To address this finding in dynamic conditions, we examined MMS-treated cells as a 188 model for stalled replication forks. Previously, we showed that MMS induces a dramatic 189 increase in RPA and Rad52 foci relative to other genotoxins (Ranatunga,N. S.,& Forsburg,S. 190 L. 2016). We observe substantial recruitment of Rad52-mCherry and brief, partial co-localization 191 of Pku70. The Pku70 signal, largely in 1-2 foci, appears after Rad52 and disappears before 192 Rad52 is resolved. Further molecular work will be required to determine what this signal 193 represents. 194 It is likely that Ku foci will define distinct structures associated with particular forms of 195 replication stress. For example, in a recent study, we showed that a mutant mcm4-dg with a 196 defect in the MCM helicase accumulates Ku foci (Kim, S. M., & Forsburg, S. L. 2020). This 197 accumulation can be reversed by activation of the Mus81 resolvase. Mus81 is essential for 198 viability in pku80∆ brc1∆ mutants (Sánchez, A., & Russell, P. 2015), indicating a collaboration 199 between Ku and Mus81 in response to replication stress. Our Pku70-citrine fusion will be a key 200 reagent in dissecting this and other activities. 201

202
Cell growth and physiology 203 Fission yeast strains are described in Table 1, and were grown as in (Sabatinos et al., 2012). 204 205
This study