Fig. 3
Fork speed and Pif1 induce flapped molecules, gapped forks, and reversed forks in Dna2-depleted cells. a The indicated CL-dna2 (Tc-dna2-AID) cells depleted (+auxin/tetracycline) for Dna2 in G1 or non-depleted (untreated) were released into S phase and RIs were analysed by TEM at 100 min from S-phase initiation (see also figure 1a for the conditions of this experiment). The chart reports the percentages and standard deviations of RIs found in the presence or in the absence of Dna2 (results are from three independent experiments, n indicates the total number of molecules analysed). *P < 0.05 by two-tailed t-test. Distributions of the lengths of the ssDNA tails in flapped molecules and ssDNA gaps in gapped forks in Dna2-depleted cells are also shown. The mean of the length of the ssDNA tails in flapped molecules or ssDNA gaps in gapped forks is reported for each chart. Grey arrows indicate abnormal RIs accumulated after a single S phase without Dna2. A representative TEM picture is shown for each type of RI found with a schematic representation of the molecule with dsDNA in black and ssDNA in red. Black scales bars of 360 nm, which correspond to 1 kilobase of DNA, are reported on each picture. b, c. As in (a) but percentages of RIs and distribution of the lengths of the ssDNA tails in flapped molecules (results from two independent experiments) are reported for tc-dna2-AID cells after a single S phase without Dna2 (auxin/tetracycline) in the absence or presence of 25 mM of HU (b), or at normal (28 °C) or low temperature (20 °C) (c). d RI analysis as in (b, c) in dna2-AID, dna2-AID pif1-m2 cells depleted for Dna2 (auxin) grown at 28 °C. b–d Grey arrows indicate suppression of accumulation of aberrant RIs in the indicated conditions and genetic backgrounds (results from two independent experiments)
