9592 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 7. Comparison of wild-type and fml1 mutant strains in the plasmid gap repair assay. (A) Schematic showing the repair of a double-strand gap in ade6 on a plasmid by HR with ade6-M26 on chromosome III. The M26 mutation is indicated by the ﬁlled circle. (B) Histogram showing the mean relative transformation efﬁciency (TE) of cut versus uncut plasmid in strains MCW1193, MCW4893, MCW2096, MCW4894 and MCW4895. (C) Histogram showing the percentage of Ura+ transformants that are Ade+ recombinants in the same strains as in (B). (D) Histogram showing the percentage of Ade+ recombinants that are crossovers in the same strains as in (B). Error bars are the standard deviations about the mean. integrated into the chromosome (Figure 7D). Intriguingly when fml1K99R and fml1D196N mutants were tested in the same assay an even greater proportion ( 50%) of Ade+ recombinants were found to be crossovers (Figure 7D). This difference is not due to the linked natMX4 marker as a fml1+::natMX4 strain behaves like the parental wild-type strain in this assay (Figure 7B–D). Altogether these data show that an ability to hydrolyse ATP is critical for Fml1’s role in promoting crossover avoidance during mitotic DSB repair. Moreover, similar to our ﬁnding with the genotoxin sensitivity assay, both point mutants appear to retain some function in vivo, which in this case impedes alternative non-crossover pathways yet is seemingly per- missive for the crossover pathway(s). fml1K99R and fml1D196N are deﬁcient for promoting gene conversion at stalled replication forks Fml1 promotes Rad51-dependent gene conversion between a direct repeat of ade6- mutant heteroalleles es- pecially when a RTS1 polar replication fork barrier (RFB) is positioned between the repeats to stall fork progression (15). On the basis of this observation and Fml1’s capabilities in vitro, we have proposed that Fml1 promotes recombination at stalled replication forks by catalysing their reversal, which generates a substrate for recombinases to act on (15). If true then both Fml1K99R and Fml1D196N, which are totally deﬁcient in catalysing fork reversal in vitro, should be unable to promote recom- bination in vivo. To test this we compared the fml1K99R and fml1D196N strains to wild-type control and fml1 strains using the RTS1-induced direct repeat recombin- ation assay shown in Figure 8A. In this assay, recombin- ation between the two ade6- genes is induced by replication fork stalling at RTS1 and gives rise to two classes of Ade+ recombinant–conversion-types, which retain an intervening his3+ gene, and deletion-types, which lose it. In the fml1+ strain, the frequency of conver- sion- and deletion-types are both in the range of 120–150 10 4, and in the fml1+::natMX4 strain, they are slightly higher at 200 10 4 (Figure 8B and C). Both fml1 and point mutants yield a similar level of deletion types as the fml1+ strain (Figure 8C). However, all three mutants show a similar 10-fold decrease in the frequency of conversion-types compared to the