Fig. 1: Mutating a Pol2 catalytic core domain impairs replication initiation and progression.

a Sequence alignment of POPS and adjacent regions in replicative polymerases. Sequences of POPS are boxed blue for the budding yeast Pol2 (ScPol2) and human POLE (hsPOLE) and absent in the catalytic subunits of DNA polymerase α (ScPol1, HsPolA) and δ (ScPol3, HsPolD). Regions adjacent to POPS, including the PALM domain, share homology among all replicative polymerases. Asterisks and dots label conserved and similar residues, respectively. Triangles highlight mutations in pol2-REL. b pol2-REL cells exhibit growth impairment. Tenfold serial dilutions of mutant and wild-type (WT) cells in biological replicates were spotted on plates and grown at the indicated temperatures. c Flow cytometry profiles suggest replication defects in pol2-REL cells. G1 synchrony was achieved by alpha-factor treatment of asynchronous culture (Asyn) at 24 °C. Flow cytometry monitored cellular DNA content upon release from G1 arrest into cycling at either 24 °C or 37 °C. d A meta-analysis of relative DNA copy numbers based on genome-sequencing results. Wild-type and pol2-REL cells were examined at 30’ and 40’ post G1-release at 24 °C as in panel c. Twenty kilo-bases from either side of early origins (n = 97, left) or late origins (n = 174, right) were averaged for copy numbers and plotted against relative positions of origins. The dotted line indicates the mid-point of origins. e Two-dimensional (2D) gel analyses reveal defective replication initiation and progression in pol2-REL cells. Samples from panel d were tested and 2D gel results for ~6 kb region containing the late origin ARS1212 or the early origin ARS305 are shown. The mid-point localization of the origins (diamonds) in the restriction fragments is shown on the side. Blue arrows signify the Y-shaped replication intermediates (RIs) in pol2-REL cells and black arrows label bubble-shaped RIs. Quantification of bubble- and Y-shaped RIs in WT and pol2-REL cells are shown at the bottom. For the former, the level of bubble-shaped RIs in WT at 20’ (for ARS305) or 30’ (for ARS1212) was set at 1. Delayed appearance of bubbled RIs and increased levels of Y-shaped RIs in pol2-REL cells were reproducibly detected in multiple trials using additional spore isolates. Signals of the bubble structures were normalized to 1N DNA to derive the percentage of bubble structures of all time points. Source data are provided as a Source data file.