Supplemental material
Experimental procedure for in vivo psoralen-DNA crosslinking of Physarum replication intermediates.
Preparation of isolated nuclei from a synchronous macroplasmodium harvested 15 min after the onset of S phase was done as previously described (Benard et al., 2001). Psoralen cross-link experiments were performed as in Liberi et al., 2006, using the same DNA-psoralen ratio. The sample was half embedded in agarose plug and half submitted to psoralen-DNA crosslinking. Briefly, isolated nuclei were resuspended in 2.5 ml of homogenization medium, kept on ice, incubated for 5 min with 25mg of psoralen (Trioxalen, Sigma), and exposed for 10 min to a 366 nm mercury-tungsten light. Incubation and illumination were repeated 4 times. Isolated nuclei were then rinsed in homogenization buffer, centrifuged and embedded in agarose plug (Benard et al., 2001). Cross-link efficiency was checked by running DNA onto an alkaline agarose gel (Benard et al., 2007). Plugs of 10mg of DNA were then digested with EcoRI and loaded on agarose gels for 2D gel experiments.
Bénard M, Maric C, Pierron G (2001) DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum. Mol Cell 7:971-980
Bénard M, Maric C, Pierron G (2007) Low rate of replication fork progression lengthens the replication timing of a locus containing an early firing origin. Nucl Acids Res 35: 5763-5774
Liberi G, Cotta-Ramusino C, Lopes M, Sogo J, Conti C, Bensimon A, Foiani M (2006) Methods to study replication fork collapse in budding yeast. Methods Enzymol 409: 442-462
Supplemental Figures and Legends
Fig. S1 2D-gel analysis of Physarum replication intermediates after in vivo psoralen-DNA crosslinking.
DNA samples taken 15 min after the onset of S phase and submitted or not to psoralen cross-link, were run on an alkaline agarose gel to validate the crosslink efficiency and then digested by EcoRI, processed by 2D gel and probed as in Fig. 1 for the actin ardC locus.
Fig. S2 Quantitations of Y arcs accumulations at the ardC locus in macroplasmodia in the presence of 50mM of HU.
Some 2D-gels from the Fig. 1a and the Fig. 2a are shown in a and b respectively. Quantitations of replication intermediates of Y arcs are indicated as a percentage of all the replication intermediates present on the 2D-gels. More precise quantitations of the different spots of the Y arcs, showing accumulations of replication intermediates, are given inside the cartoons below each frame as a percentage of the total Y arc
Fig. S3 Persistence of replication initiation induced by HU in microplasmodia.
Kinetics of a long lasting 10mM HU treatment for the EcoRI restriction fragment of the ardC actin promoter (a), and the HindIII restriction fragment from the proP profilin promoter (b), during the growing phase of the microplasmodia are shown from 4 to 16h, together with the control 2D-gels. The proP probe was the one already used in Maric et al., 2010
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