Figure 6.

Ulp2 Supports Efficient Replication Onset by Safeguarding MonoSUMOylated DDK Engaged in Replication

(A and B) Synthetic lethality of dbf4-1 ulp2Δ and cdc7-4 ulp2Δ cells at permissive temperatures for dbf4-1 and cdc7-4 single mutants is suppressed by a lysine-less SUMO variant (smt3-KRall).

(C) Reduced DDK-mediated Mcm4 phosphorylation in ulp2Δ cells is suppressed by deleting SLX5. Shown is IP of Mcm4^13MYC from exponentially growing WT, slx5Δ, ulp2Δ, and ulp2Δ slx5Δ cells. The specificity of the anti-Mcm4-phospho-S82-D83 antibody was evaluated by lambda phosphatase treatment (λ) with or without phosphatase inhibitors (Inh).

(D) The SUMOylation-defective ddk-KR mutant rescues reduced DDK-dependent Mcm4 phosphorylation in ulp2Δ cells; as in (C), but Mcm4^13MYC IP from WT, ddk-KR, ulp2Δ, and ulp2Δ ddk-KR cells.

(E) The decrease in BrdU incorporation in ulp2Δ cells is suppressed by ddk-KR, similar to slx5Δ. Shown is BrdU IP-on-chip analysis of cells released into S phase in the presence of 0.2 M HU and BrdU for 90 min after G1 arrest.

(F) The S phase progression defect in ulp2Δ cells is suppressed by ddk-KR. Exponentially growing WT, ddk-KR, ulp2Δ, and ulp2Δ ddk-KR cells (cycl) arrested in G1 phase by α-factor were released into yeast extract-peptone-dextrose (YPD) media at 25°C, and samples were taken every 5 min for FACS.

See also Figure S6.