9586 Nucleic Acids Research, 2012, Vol. 40, No. 19 Table 1. Schizosaccharomyces pombe strains used in this study Strain MCW1221 MCW2080 MCW4581 MCW4778 MCW4779 MCW1193 MCW2096 MCW4893 MCW4894 MCW4895 MCW4713 MCW3061 MCW4797 MCW4780 MCW4801 MCW4836 MCW6137 MCW6138 Relevant genotype h+ ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1 ::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1+::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1D196N::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1K99R::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M26 h+ fml1 ::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M26 h+ fml1+::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M26 h+ fml1D196N::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M26 h+ fml1K99R::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M26 h+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 int::pUC8/his3+/RTS1 site A orientation 2/ade6-L469 h+ fml1 ::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 int::pUC8/his3+/RTS1 site A orientation 2/ade6-L469 h+ fml1+::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 int::pUC8/his3+/RTS1 site A orientation 2/ade6-L469 h+ fml1K99R::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 int::pUC8/his3+/RTS1 site A orientation 2/ade6-L469 h+ fml1D196N::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 int::pUC8/his3+/RTS1 site A orientation 2/ade6-L469 h+ fml1+-GFP::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1D196N-GFP::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 h+ fml1K99R-GFP::natMX4 ura4-D18 leu1-32 his3-D1 arg3-D4 Source Laboratory strain (15) This study This study This study Laboratory strain (15) This study This study This study Laboratory strain (15) This study This study This study This study This study This study Microscopy Cells from an overnight culture in YES were harvested, washed in water, re-suspended in fresh YES and grown for 3 h at 30 C before addition of 0.03% methyl methane sulphonate (MMS). Incubation was continued for a further 4 h before the cells were harvested and washed to remove the MMS. They were then re-suspended in fresh YES and allowed to recover at 30 C. Samples were taken at hourly intervals and ﬁxed with 70% ethanol for subse- quent microscopy. The ﬁxed cells were stained with DAPI and analysed using an Olympus BX50 epiﬂuorescence microscope equipped with the appropriate ﬁlter sets to detect blue and green ﬂuorescence (Chroma Technology Corp., VT). Black and white images were acquired with a CoolSNAP HQ CCD camera (Photometrics, AZ) controlled by MetaMorph software (v7.7.3.0, Molecular Devices Inc., CA). Recombination assays The direct repeat recombination assay and plasmid gap repair assay were performed as described (15,26,27). Two sample t-tests were used to determine the statistical signiﬁcance of differences in recombination values between strains. Protein puriﬁcation Wild-type and mutant Fml1 C were puriﬁed as described (15). Amounts of protein are expressed in moles of monomer, and were estimated using a Bio-Rad protein assay kit with bovine serum albumin (BSA) as the standard. ATPase assay Reaction mixtures (20 ml) contained 5 nM wild-type or mutant Fml1 C protein, 2.5 mM MgCl2 and 200 nM PhiX174 circular single-stranded (ss) DNA (New England Biolabs) in buffer (25 mM Tris–HCl, pH 8.0, 1 mM dithiothreitol (DTT), 100 mg/ml BSA, 6% glycerol). Reactions were started by the addition of 5 mM ATP and then incubated at 37 C for 30 min before being stopped by the addition of 5 ml of 200 mM ethylenediaminetetraacetic acid (EDTA). The extent of ATP hydrolysis was determined by measuring the amount of inorganic phos- phate that had been released into the reaction mixture using a malachite green colourimetric assay based on that developed by Baykov et al. (28). In brief malachite green solution (0.5 mM malachite green oxalate, 0.1 N HCl) was mixed with ammonium molybdate solution (34 mM ammonium molybdate tetrahydrate, 4 N HCl) in a 3:1 ratio and ﬁltered through two 0.22 mM ﬁlters to make the colourmetric reagent, which was stored in the dark; 700 ml of the colourmetric reagent was added to the stopped reaction mixture and incubated for 1 min at room temperature. The development of colour was then stopped by addition of 200 ml 34% (w/v) sodium citrate followed by mixing using a vortex mixer. Absorbance of the mixture at 610 nm was then measured and the amount of inorganic phosphate determined by reference to known standards. DNA substrates The 32P-labelled DNA substrates in Figure 2, Supple- mentary Figures S1 and S3 were made by annealing the following combination of oligonucleotides: X-12, oligo- nucleotides 1–4; linear dsDNA, oligonucleotides 2 and oMW96; X0, oligonucleotides 2, 5, 6 and 7; fork, oligo- nucleotides 2, 5 and 11. Oligonucleotides 1–7 and 11, and the method of substrate preparation have been described (29,30). The sequence of oligonucleotide oMW96 is 50-CA ACGTCATAGACGATTACATTGCTAGGACATCTT TGCCCACGTTGACCC-30. Oligonucleotides were supplied by Sigma-Genosys Ltd. and were puriﬁed as described (31). The model replication fork in Figure 3