Figure 3. The catalysis of fork regression by wild-type and mutant Fml1 Cs. (A) Schematic showing the model replication fork and its regression catalysed by Fml1. Letters A, E, F and S are the cleavage sites for AvrII, EcoRI, AﬂIII and ScaI, respectively. The asterisk indicates the position of the 50-end 32P-label on what is the equivalent of the nascent lagging strand. (B) Comparison of fork regression catalysed by Fml1 C (lanes a–d), Fml1 CK99R (lanes e–h) and Fml1 CD196N (lanes i–l). See ‘Materials and Methods’ section for further details. activity might not be wholly dependent on an ability to hydrolyse ATP (17). It was therefore important that Fml1 CK99R and Fml1 CD196N were tested to see whether they similarly retained an ability to catalyse this reaction. For this we used a model replication fork, which when regressed contains an extruded duplex DNA arm with 50-end radiolabel (Figure 3A). The extent of fork reversal can be monitored by the ability to cleave the extruded arm with different restriction enzymes. As expected wild-type Fml1 C readily catalyses fork reversal with branch point migration occurring over dis- tances of up to 1813 bp or more (Figure 3B, lanes a–d). In contrast, both Fml1 CK99R and Fml1 CD196N exhibit no detectable fork reversal activity even over relatively short distances of 36 bp (Figure 3B; lanes e–h and i–l). This deﬁciency in catalysing fork regression is not caused by an inability to bind fork DNA because both mutant proteins display similar binding as wild-type Fml1 C to Nucleic Acids Research, 2012, Vol. 40, No. 19 9589 Figure 4. Spot assays comparing the genotoxin sensitivities of strains MCW1221, MCW4581, MCW2080, MCW4778 and MCW4779. a model replication fork (Supplementary Figure S3). Together these data indicate that the catalysis of fork reversal by Fml1 C in vitro depends on its ability to hydrolyse ATP.