Contrasting Roles of Checkpoint Proteins as Recombination Modulators at Fob1-Ter Complexes with or without Fork Arrest

Contribution of the interplay between TOF1 and RRM3 to plasmid integration and excision. (A) Autoradiogram of a representative Southern blot showing the effect of tof1Δ rrm3Δ double deletions on plasmid integration (pBB-Hyg) in comparison with the single deletions, all in a common sir2Δ background after three and six serial streakings and growth in the selective medium. (B) Effects of single and double deletions (tof1Δ, rrm3Δ, and tof1Δ rrm3Δ) on pBB3NTS (URA3) plasmid excision. (C) Autoradiograms of 2D gels showing replication fork arrest at Ter in different strains; the arrows show the termination spots generated by fork arrest at the two closely spaced Ter1 and Ter2 sites. WT, wild type.

Impact of deletions of genes involved in HR and NHEJ on plasmid integration and excision. (A) Autoradiogram of a representative Southern blot of nicked DNA showing the status of intracellular plasmid DNA pBB3NTS (URA3) in isogenic strains containing various deletions. The lanes are self-explanatory. (B) Plasmid excision kinetics showing the impact of various deletions of recombination genes on the excision of pBB3NTS (URA3). (C) Effect of deletions of two structure-specific endonucleases (slx4Δ, mus81Δ, and slx4Δ mus81Δ) on plasmid pBB3NTS (URA3) integration; the lanes are self-explanatory. WT, wild type.

Impact of deletions of genes encoding checkpoint proteins and recombination proteins on HOT1 activity. (A) Schematic diagram showing the HOT1 assay; the E elements (containing the Fob1 binding site) and the I elements are shown; recombination between the flanking his4 genes excises URA3, which has no ARS and therefore is eliminated from the cells. (B) HOT1 activity in the absence of checkpoint adapter proteins. (C) HOT1 activity in the absence of MEC1 and RAD53. (D) HOT1 activity in different strains with individual deletions of the various members of the RAD52 epistasis group. WT, wild type.