displacement (D)-loops that are formed by Rad51- mediated strand invasion (14–18). The latter activity shows how they could be capable of promoting a form of DSB repair called synthesis dependent strand annealing (SDSA), which involves D-loop dissociation and generates solely non-crossover recombinants (1). A failure of SDSA can lead to an increase in crossover recombinants through the cleavage of D-loops and HJs by junction-speciﬁc endo- nucleases (19). In addition to an ability to unwind D-loops, FANCM family members are also adept at catalysing replication fork reversal (15,16,18,20). This reaction involves the un- winding of both nascent strands at a stalled replication fork followed by their annealing and the re-annealing of the parental strands. This activity may serve several func- tions including: (i) facilitating DNA repair by providing space for the excision of strand lesions that block fork progression; (ii) providing a means of tolerating DNA damage by enabling the DNA polymerase to switch from the damaged template strand to the undamaged equivalent nascent strand and thereby bypass the lesion and (iii) promoting recombination-dependent replication restart by generating a double-stranded (ds) DNA end, which can act as an entry point for recombinases. Consistent with a role in catalysing fork reversal Fml1 has been shown to promote Rad51-dependent recombin- ation at a blocked replication fork in vivo (15,20). The ability of various FANCM family members to unwind D-loops and reverse replication forks in vitro depends on a supply of hydrolysable ATP (1). Mutations that impair ATP hydrolysis in FANCM and Mph1 have been shown to confer deﬁciencies in DNA repair and crossover avoidance in vivo (12,14). However, in some cases, biological activity is not destroyed presum- ably because FANCM can also act as a molecular scaffold for the recruitment of proteins (7,8,12,21). Here, we deter- mine whether Fml1’s roles in DNA repair, crossover avoidance and processing stalled replication forks are de- pendent on its ability to hydrolyse ATP. MATERIALS AND METHODS Schizosaccharomyces pombe strains and plasmids Schizosaccharomyces pombe strains are listed in Table 1. The fml1+::natMX4 strain was made by gene targeting using a derivative of pAG25 (22), which was constructed as follows. First, the ADH1 terminator from pFA6a- GST-kanMX6 was sub-cloned as a AscI-BglII fragment into pAG25 to make pMW756. A 300 bp SacI-EcoRI fragment from the fml1 30-UTR region was then sub-cloned from pMW778 into pMW756 to make pSN13. A SalI-SmaI fragment encompassing the entire fml1+ open reading frame (ORF) was then sub-cloned from pMW848 (see below) into the SalI and PacI (blunt-ended by Klenow) sites in pSN13 to make pSN14. The gene-targeting fragment was excised from pSN14 by digestion with SalI and partial digestion with XbaI. Derivatives of pSN14, in which either an A296G (pSN16) or G586A (pSN15) point mutation was introduced into fml1+ using QuikChange XL site-directed Nucleic Acids Research, 2012, Vol. 40, No. 19 9585 mutagenesis (Agilent Technologies, CA), were used to make the fml1K99R::natMX4 and fml1D196N::natMX4 strains. The fml1+-GFP::natMX4 strain was made by gene targeting using a derivative of pFA6a-GFP(S65T)- kanMX6 (23), which was constructed as follows. First, a BglII-EcoRV fragment containing the natMX4 and fml1 30-UTR from pSN13 was sub-cloned into pFA6a- GFP(S65T)-kanMX6. The 30-end of the fml1 ORF was then ampliﬁed from genomic DNA using oMW1004 (50-AGAAAGGCTGCTGGAATGTC-30) and oMW 1005 (50-TATTTAATTAAAATCAGCATTCCTTTCAT ACG-30) and cloned as a NdeI-PacI fragment into this plasmid to make pJBB31. The gene-targeting fragment was excised from pJBB31 by digestion with NdeI and XbaI. To construct the fml1K99R-GFP::natMX4 and fml1D196N-GFP::natMX4 strains, fml1K99R::natMX4 and fml1D196N::natMX4 strains were ﬁrst converted to fml1K99R::hphMX4 and fml1D196N::hphMX4, respectively, by gene targeting using a BamHI-EcoRV fragment from pAG32 containing the hygromycin resistance gene. The resultant strains were then subjected to gene targeting using the NdeI and XbaI fragment from pJBB31 as described earlier. The plasmid for expressing His-tagged Fml1 C in Escherichia coli was pSN3 and has been described previously (15). Expression plasmids for His-tagged Fml1 CK99R (pSN5) and Fml1 CD196N (pSN4) were derived from pSN3 by site-directed mutagen- esis as described earlier. pREP41-Fml1 C (pMW856) has been described (15). pREP41-Fml1 CK99R (pSN22) and pREP41 CD196N (pSN21) were derived from pMW856 by site-directed mutagenesis as described earlier. pREP41-Fml1 (pMW848) was constructed by amplifying full-length fml1 from genomic DNA, using primers oMW873 (50-TATTAGTCGACATGTCCGATGATTCT TTTAG-30) and oMW874 (50-TATTCCCGGGATTCTC TAAATCAGCATTCC-30) and cloning it as a SalI-SmaI fragment into pREP41 (24). pREP41-Fml1K99R (pSN23) and pREP41D196N (pSN24) were derived from pMW848 by site-directed mutagenesis as described earlier. All plasmids were veriﬁed by DNA sequencing. Media and genetic methods Media and genetic methods followed standard protocols (25). The complete and minimal media were yeast extract with supplements (YES) and Edinburgh minimal medium plus 3.7 mg/ml sodium glutamate (EMMG) plus appropri- ate amino acids (0.25 mg/ml), respectively. Low adenine media (YELA) was supplemented with 0.01 mg/ml adenine. Ade+ recombinants were selected on YES lacking adenine and supplemented with 0.2 mg/ml guanine to prevent uptake of residual adenine. Spot assays Exponentially growing cells from liquid cultures were har- vested, washed and re-suspended in water at a density of 1 107–1 103 cells/ml. Aliquots (10 ml) of the cell suspen- sions were spotted onto agar plates containing genotoxins as indicated. For UV, plates were irradiated using a Stratalinker (Stratagene). Plates were photographed after 4 days growth at 30 C unless otherwise indicated.