9590 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 5. Spot assays comparing the MMS sensitivities of wild-type (MCW1221) and fml1 (MSW2080) strains carrying the indicated plasmids. Spot assays in A were photographed after 4 days growth at 30 C, whereas those in B were photographed after 5 days. Genotoxin sensitivity of fml1K99R and fml1D196N mutants Having established that Fml1 CK99R and Fml1 CD196N are deﬁcient in ATP hydrolysis, junction unwinding and junction branch migration in vitro, we next investigated their function in vivo. To do this, the endogenous fml1 gene was replaced with the mutant versions linked to a downstream natMX4 marker (fml1K99R::natMX4 and fml1D196N::natMX4). To control for any effects of the marker, a strain containing fml1+ with natMX4 down- stream was also constructed (fml1+::natMX4). We have previously shown that a strain in which fml1+ is deleted (fml1 ) exhibits hypersensitivity to cisplatin and MMS (15). To assess the impact of the K99R and D196N mutations on genotoxin sensitivity, we performed a spot assay in which serial dilutions of the relevant strains were spotted onto complete media with or without genotoxin (Figure 4). Consistent with our previous data, the fml1 mutant exhibits hypersensitivity to cisplatin and MMS but is as resistant as wild-type to hydroxyurea (HU) and the topoisomerase I poison camptothecin (CPT). We also observed a slight hypersensitivity to high doses of ultra- violet light (UV), which had been missed in our previous study. Both point mutants are similarly sensitive to cis- platin and UV as the deletion strain and slightly more sensitive to MMS (Figure 4). Intriguingly, and unlike the deletion mutant, they are also modestly hypersensitive to CPT. These sensitivities can be directly attributed to the point mutations in fml1 rather than the linked natMX4 marker because the fml1+::natMX4 strain exhibits no hypersensitivity to any of the genotoxins used here (Figure 4). In fact it is slightly more resistant than the wild-type strain from which it was derived. The reason for this enhanced resistance is currently unclear. Overall these data show that Fml1’s ATPase activity is essential for its ability to promote DNA damage repair/tolerance. The heightened sensitivity of the ATPase-deﬁcient mutants suggests that they retain the ability to bind their intended substrates in vivo and in so-doing block other alternative repair/tolerance pathways.