Fig. 4: Loss of H3K4 methylation and transcription progression prevents defective replication fork formation in rad53 mutants.

a Schematic representation of the signal profile for 2D gel analyses. b A schematic representation of ARS305 for 2D gel analysis is shown. c 2D gel analysis after in vivo psoralen crosslinking of the EcoRI-PstI-digested (blue) or SacI-digested (red) fragments of the ARS305 region in RAD53-H3, RAD53-H3K4A, rad53-K227A-H3, and rad53-K227A-H3K4A cells released from G1 into 25 mM HU at the indicated time-points. FACS profiles show the cellular DNA content during the experiments. The purple arrows indicate spike signals corresponding to aberrant DNA intermediates that accumulated in HU in rad53-K227A mutants. d A schematic representation of the inducible MET25 promoter driving the 3HA-LRE1 gene with a head-on orientation to ARS305. Met (+) indicates S. C. medium, which suppresses 3HA-LRE1 by inactivating the MET25 promoter. Met (−) indicates S. C. minus methionine medium, which turns on 3HA-LRE1 by the MET25 promoter. e 2D gel analysis of the EcoRI-PstI-digested fragments on ARS305 region in RAD53-H3 and RAD53-H3K4A cells released from G1 into 25 mM HU at the indicated time-points in Met (−) medium. f As in e, 2D gel analysis of rad53-K227A-H3 and rad53-K227A-H3K4A cells in Met (−) or Met (+) medium. g 2D analysis of SacI-digested replication intermediates from rad53-K227A-H3 and rad53-K227A-H3K4A cells with either HO (head on)-TRC or CD (co-directional)-TRC between 3HA-LRE1 transcription and fork originated from ARS305 following G1 synchronization and subsequent release into 25 mM HU for 2 h. Met (−) and Met (+) as in (a–c). A schematic representation of putative replication fork intermediates represented by the 2D-gel signal shown in HO-TRC (red box) and CD-TRC (blue box). h 2D gel analysis of HindIII-PstI-digested DNA fragments from rad53-K227A and set1Δ, bre2Δ, or spp1Δ on the rad53-K227A background to monitor replication intermediates at ARS1211 after G1 release into HU treatment for 2 h.