Nucleic Acids Research, 2012, Vol. 40, No. 19 9593 Figure 8. RTS1-induced direct repeat recombination in wild-type and fml1 mutant strains. (A) Schematic showing the direct repeat recombination substrate on chromosome III plus the two classes of Ade+ recombinant product. (B) Ade+ His+ (conversion-types) recombinant frequencies for strains MCW4713, MCW4797, MCW3061, MCW4801 and MCW4780. (C) Ade+ His (deletion-types) recombinant frequencies for the same strains as in (B). In both (B) and (C), the error bars represent the standard deviations about the mean. fml1+ strain (Figure 8B). These data show that Fml1’s ATPase activity, which is integral to its ability to catalyse replication fork reversal, is essential for promoting Rad51-dependent recombination at stalled rep- lication forks. DISCUSSION We have shown that Fml1’s ability to hydrolyse ATP is essential for its function in promoting repair/tolerance of DNA damage, crossover avoidance during DSB repair and Rad51-dependent gene conversion at a blocked repli- cation fork. As Fml1 uses ATP hydrolysis to fuel its un- winding and branch migration of junction DNA it is likely that these activities are central to its aforementioned roles. Importantly mutants that are defective in ATP hydrolysis display wild-type levels of junction DNA binding in vitro, and therefore simply binding to its proposed substrates in vivo is not sufﬁcient for Fml1 to perform its DNA repair and recombinogenic functions. Similar to our ﬁndings, studies of FANCM in human cells have shown that a K117R mutation in helicase motif 1, which is equivalent to K99R in Fml1, results in a deﬁciency in cisplatin resistance (7,21). Interestingly this mutation does not impair FANCM’s ability to promote the monoubiquitination and localization into DNA repair foci of FANCD2 (7,21). These data indicate that FANCM can serve at least two functions in promoting DNA repair: (i) it binds DNA and thereby can act as a scaffold for the recruitment of the FA core complex to chromatin; and (ii) it can use the energy from hydrolysing ATP to branch migrate and unwind junction DNA, which can potentially promote ICL repair in various ways as dis- cussed earlier. Our observation that ATPase-dead Fml1