Replication factor C complexes play unique pro- and anti-establishment roles in sister chromatid cohesion

Abstract

Recent studies have lead to a rapid expansion of sister chromatid cohesion pathways. Of particular interest is the growth in classifications of anti-establishment factors-now including those that are cohesin-associated (Rad61/WAPL and Pds5) or DNA replication fork-associated (Elg1-RFC). In this study, we show that the two classes of anti-establishment complexes are indistinguishable when challenged both genetically and functionally. These findings suggest that both classes function in a singular pathway that is centered on Ctf7/Eco1 (herein termed Ctf7) regulation. The anti-establishment activity of Elg1-RFC complex is particular intriguing given that an alternate Ctf18-RFC complex exhibits robust pro-establishment activity. Here, we provide several lines of evidence, including the use of Ctf7 bypass suppressors, indicating that these activities are not simply antagonistic. Moreover, the results suggest that Ctf18-RFC is capable of promoting sister chromatid pairing reactions independent of Ctf7. The combination of these studies suggest a new model of sister chromatid pairing regulation.

Figure 1. SMC3 acetylation mimetic provides no growth benefit to ctf18 mutant cells.

10 fold serial dilutions of smc3^Q and ctf18 single mutant cells and three independent isolates of ctf18 smc3^Q double mutant cells. Colony growth shown for cells on rich medium plates grown at 18°C, 23°C, 30°C, and 37°C for number of days indicated. Strains shown include YMM872, YMM873, YMM874, YMM875 and YMM 876.

Figure 2. rad61 deletion provides limited bypass of ctf7 mutant cell lethality.

10 fold serial dilutions of wild type, rad61 and ctf7-203 single mutant cells and also ctf7 rad61 double mutant cells. Colony growth shown for cells on rich medium plates grown at 18°C, 23°C, 30°C, and 37°C for number of days indicated. Strains shown include YBS255, YMM808, YBS514 and YMM828.

Figure 3. Neither rad61 nor elg1 deletion, nor the combination, rescue ctf18 deficiency in ctf7 mutant cells.

10 fold serial dilutions of ctf7 rad61 double, ctf7 rad61 elg1 and ctf7 rad61 ctf18 triple mutant cells and also ctf7 rad61 ctf18 elg1 quadruple mutant cells. Colony growth shown for cells on rich medium plates grown at 23°C, 30°C and 37°C for the number of days indicated. Strains shown include YMM828, YMM829, YMM821, YMM823, YMM820, YMM827, YMM822 and YMM824.

Figure 4. rad61 ctf7 ctf18 triple mutant cells progress normally through S phase.

DNA profiles of rad61 and ctf18 single mutant cells and rad61 ctf18 and rad61 ctf7 and also rad61 ctf7 ctf18 triple mutant strains during log phase growth (Log), synchronized in G₁ (α-Factor) at 30°C and then released into fresh medium at 37°C. Time points after release into fresh medium indicated. Strains shown include YMM808, YBS1160, YMM813, YMM829 and YMM825.

Figure 5. elg1 deletion and smc3 acetylmimetic alleles exhibit similar effects on ctf7 mutant cells.

10 fold serial dilutions of ctf7-203 single mutant cells, ctf7-203 elg1 and ctf7-203 smc3^Q double mutant cells and four independent isolates of ctf7-203 smc3^Q elg1 triple mutant cells. Colony growth shown for cells on rich medium plates grown at 18°C, 23°C, 30°C and 37°C for the number of days indicated. Revertant (R) triple mutant cell shown. Strains shown include YMM865, YMM866, YMM867, YMM869, YMM870 and YMM871.

Figure 6. rad61 deletion fails to provide growth benefit to either elg1 or ctf18 mutant cells.

10 fold serial dilutions of rad61, ctf18 and elg1 single mutant cells, rad61 ctf18, rad61 elg1 and ctf18 elg1 double mutant cells and also two independent isolates of rad61 ctf18 elg1 triple mutant cells. Colony growth shown for cells on rich medium plates grown at 18°C, 23°C, 30°C, and 37°C for number of days indicated. Strains shown include YMM808, YBS1159, YMM207, YMM812, YMM818, YMM298, YMM816 and YMM817.

Figure 7. Cohesion assays comparing wildtype, rad61 and elg1 single mutant cells and rad61 elg1 double mutant cells.

Top left: Differential Interference Contrast (DIC) microscopy epi-fluorescence microscopy images highlight cell morphology and co-localization of DNA (DAPI) and sister chromatid loci (GFP). Bottom: DNA content profiles obtained by flow cytometry. Top right: Quantification of sister chromatid pairing defects (see Materials and Methods). Error bars represent max and min of each trial. Strains shown include YMM334, YMM985 and YMM988.

Figure 8. Expression of smc3 acetylmimetic in pol30-104 mutant cells.

10 fold serial dilutions of smc3^Q and pol30-104 single mutant cells and three independent isolates of smc3^Q pol30-104 double mutant cells. Colony growth shown for cells on rich medium plates grown at 18°C, 23°C, 30°C, and 37°C for number of days indicated. Strains shown include YMM890, YMM891, YMM892, YMM893 and YMM894.

Figure 9. Effects of POL30 (PCNA) over-expression in ctf7-203 (ctf7) single mutant cells and ctf7-203 elg1 smc3^Q triple mutant cells.

Vector control plasmid (V) also shown. 10 fold serial dilutions of log phase growth cells plated onto selective medium shown after growth at 23°C and 30°C. Strains shown include YMM918, YMM919, YMM920 and YMM921.

Figure 10. Model of anti-establishment (Elg1-RFC) and pro-establishment (Ctf18-RFC and Ctf18-Dcc1-Ctf8) complexes.

Two pro-establishment pathways are described, one of which occurs independent of Ctf7-dependend acetylation of Smc3. Several speculative mechanisms are highlighted. In contrast, Elg1-RFC and Rad61 may function through a singular pathway. See text for details.