Over-expression of Fml1K99R and Fml1D196N sensitizes both wild-type and fml1" strains to MMS To further investigate the toxicity of Fml1K99R and Fml1D196N, we analysed the effect of their over-expression on MMS sensitivity in wild-type and fml1 mutant strains. The thiamine repressible nmt41 promoter in plasmid pREP41 was used to drive over-expression (24), and MMS sensitivity was assessed by spot assays (Figure 5). Consistent with previous data, plasmid-borne fml1+ and fml1 C efﬁciently complement the MMS sen- sitivity of a fml1 mutant when expressed from the nmt41 promoter (Figure 5A) (note that the nmt41 promoter is leaky and therefore still drives some expression even in the presence of thiamine) (15). As expected, expression of either full-length or C-terminally truncated Fml1 K99R and D196N mutants failed to rescue MMS sensi- tivity, conﬁrming that ATP hydrolysis is essential for Fml1’s role in promoting genotoxin resistance. Moreover, in the absence of thiamine (i.e. when the nmt41 promoter is de-repressed), they actually increased fml1 mutant sensitivity (Figure 5A). Over-expression of either full-length or C-terminally truncated Fml1K99R/ Fml1D196N also sensitized wild-type cells to MMS and im- portantly the degree of sensitization was slightly greater than seen in a fml1 strain carrying the empty pREP41 plasmid (Figure 5B, compare the sensitivity of strains to 0.008% MMS in the absence of thiamine). Altogether these data suggest that over-expression of ATPase-deﬁcient Fml1 (with or without its C-terminal domain) can interfere both with endogenous wild-type Fml1 and other repair/tolerance pathways in their response to MMS-induced damage. Fml1K99R-GFP and Fml1D196N-GFP form detectable nuclear foci following MMS treatment To investigate whether Fml1K99R/Fml1D196N persists at sites on DNA following MMS-induced damage, we con- structed strains that express wild-type/mutant Fml1 with a C-terminal green ﬂuorescent protein (GFP) fusion from the endogenous fml1 promoter. All three strains express similar levels of the fusion protein and exhibit the same MMS sensitivity as the equivalent strain without GFP (data not shown). Cells were exposed to MMS for 4 h and then monitored for the appearance of nuclear foci during a 6-h recovery period. In cells expressing the Fml1+-GFP fusion, only background ﬂuorescence was detected both in the absence of MMS and following MMS exposure (Figure 6B and data not shown). The same was true for cells expressing Fml1K99R/D196N-GFP in the absence of MMS; however, weak nuclear foci were detected in up to 14% of cells at the 3–6 h time points during the recovery period following MMS exposure (Figure 6A and B). In almost all these cases, only a single focus was detected in each nucleus. These data show that, in contrast to wild-type Fml1, Fml1K99R/D196N can accumu- late at sites on DNA and persist there long enough for foci to be detected. Nucleic Acids Research, 2012, Vol. 40, No. 19 9591 Figure 6. Induction of Fml1K99R-GFP and Fml1D196N-GFP foci by MMS. (A) Example of a cell with a Fml1D196N-GFP focus. DNA was stained with DAPI. (B) Percentage of cells with a nuclear GFP focus. Values are the mean of two to three independent experiments except for the Fml1D196N-GFP 5- and 6-h time points, which are from a single experiment. In each experiment >100 cells were analysed for each time point. Error bars represent the range. fml1K99R and fml1D196N mutants exhibit elevated levels of crossing over resulting from mitotic DSB repair We have shown previously that a fml1 strain exhibits high levels of crossing over in a plasmid gap repair assay (15). This assay involves transformation of a plasmid, which has been cut to generate a 153 bp double-stranded gap within a copy of ade6, into a strain containing a mutant allele of ade6 (ade6-M26) at its normal genomic locus on chromosome III. The linearized plasmid is repaired in vivo by HR, with the chromosomal ade6-M26 acting as the donor of genetic information during the reaction (Figure 7A). In wild-type cells, the majority of the repaired plasmids ( 75%) contain a fully restored ade6+ gene through gene conversion with chromosomal ade6-M26, and most of these ( 90%) are non-crossovers, where the plasmid has re-circularized (Figure 7A, C and D). A fml1 mutant exhibits similar levels of plasmid repair and Ade+ recombinants as wild- type (Figure 7B and C), but unlike wild-type >30% of the recombinants are crossovers, where the plasmid has