9594 Nucleic Acids Research, 2012, Vol. 40, No. 19 mutants exhibit no residual DNA repair capacity suggests that in S. pombe Fml1 does not play an important scaf- folding role, which is in line with the apparent absence of other FA pathway proteins. The idea that ATP hydrolysis is needed for FANCM to promote ICL repair has been questioned following the observation that a D203A mutation in helicase motif II failed to cause hypersensitivity to cisplatin in chicken DT40 cells (12). Like the K117R mutation in motif I, the D203A mutation destroys ATPase activity; however, unlike K117R it is thought not to impair ATP binding. Based on this it was proposed that FANCM’s motor activity is unnecessary for promoting DNA crosslink repair, which instead simply relies on a conformational change in FANCM brought about by binding ATP (12). However, our data show that a D196N mutation in Fml1, which is the equivalent residue as D203 in chicken FANCM, produces a mutant phenotype with respect to cisplatin sensitivity. This suggests that ATP binding on its own is not sufﬁcient in the case of the ﬁssion yeast FANCM orthologue for it to perform its role in DNA repair. One intriguing observation that we have made is that both fml1K99R and fml1D196N mutants in some cases exhibit phenotypes that are more extreme than those of the mutant. Namely they are more sensitive to MMS, exhibit hypersensitivity to CPT that is not seen with the mutant and display higher levels of crossing over in the plasmid gap repair assay. Over-expression of these Fml1 mutants also sensitizes wild-type cells to MMS to a greater degree than deleting fml1 and increases the hypersensitivity of a fml1 mutant. These data suggest that Fml1K99R and Fml1D196N retain a function in vivo that blocks alternative DNA processing pathways. For example, MMS is known to cause lesions that block rep- lication fork progression (35), and Fml1’s normal role here may be to promote lesion bypass through catalysing fork reversal (15). Presumably, the mutant proteins would bind to stalled forks, as they do in vitro, and in so doing impede other mechanisms of repair or lesion tolerance, such as TLS or those that rely on alternative fork reversal pathways. In this respect, it is worth noting that a fml1 mutant, similar to a mph1 mutant in S. cerevisiae, exhibits a hyper-mutator phenotype indicating increased usage of TLS to tolerate replication fork blocking lesions (36). Furthermore, loss of Fml1 also results in greater reliance on Rad8 for survival in the pres- ence of MMS (15). Rad8 is the orthologue of S. cerevisiae Rad5, which is a key component of the error-free branch of the post-replication repair pathway and has been proposed to catalyse fork reversal (32). Evidence that Fml1K99R/Fml1D196N might persist on DNA following MMS-induced damaged comes from our observation that both mutant proteins when fused to GFP form detectable nuclear foci following exposure to MMS, whereas wild-type Fml1 does not. However, these foci are only detected 3 h after MMS is removed, which is not consistent with Fml1K99R/Fml1D196N accumulating at replication forks when they ﬁrst stall during S-phase. Possibly Fml1 is recruited later to a subset of ‘problem’ replication forks that persist into the G2 and M phases of the cell cycle. Binding to its substrate and preventing its normal pro- cessing may also account for the increased levels of crossing over during DSB repair in fml1K99R and fml1D196N mutants. In this case, the substrate is most likely a D-loop that is formed by Rad51-mediated strand invasion. By binding to the D-loop Fml1K99R/ Fml1D196N could prevent its maturation into a double HJ, which is a substrate for the dissolution pathway that appears to be the main alternative to SDSA for avoiding crossovers (15,37). In the absence of both major non-crossover pathways, D-loops could be resolved by junction-speciﬁc endonucleases such as Mus81-Eme1 that generate crossover recombinants (15,38). Indeed, a mus81 fml1 double mutant exhibits a synthetic reduc- tion in viability and enhanced genotoxin sensitivity con- sistent with the idea that Mus81 and Fml1 function in alternative pathways for processing D-loops (15). Seemingly, the presence of Fml1K99R/Fml1D196N at the D-loop is not a barrier to Mus81-Eme1 and might even aid it by holding it in a confirmation that is amenable to cleavage. Future in vitro studies may provide insight into how these enzymes compete for the same substrate. SUPPLEMENTARY DATA Supplementary Data are available at NAR Supplementary Figures 1–3. Online: ACKNOWLEDGEMENTS The authors thank Leonard Wu (University of Oxford) for providing the plasmids for constructing the model replication fork and their lab members Fekret Osman and Sonali Bhattacharjee for providing strains, Vincent Mason for technical assistance and Fekret Osman, Alexander Lorenz and Roland Steinacher for critical reading of the manuscript. FUNDING The Wellcome Trust [090767/Z/09/Z]; K. Pathak Clarendon Scholarship from Exeter College, University of Oxford (to S.N.). Funding for open access charge: The Wellcome Trust. Conﬂict of interest statement. None declared. REFERENCES 1. Whitby,M.C. (2010) The FANCM family of DNA helicases/ translocases. DNA Repair, 9, 224–236. 2. Meetei,A.R., Medhurst,A.L., Ling,C., Xue,Y., Singh,T.R., Bier,P., Steltenpool,J., Stone,S., Dokal,I., Mathew,C.G. et al. (2005) A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nat. Genet., 37, 958–963. 3. 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