Effects of the Cell Cycle and RAD18 Overexpression on PCNA Ubiquitylation
(A) Cell-cycle dependence of PCNA modification. Cells arrested in G1, S, and G2 phase were treated with 0.02% MMS for 90 min where indicated, and modifications of ^HisPCNA, isolated under denaturing conditions, were detected by western blot. DNA contents were monitored by flow cytometry (FACS). Asynchronous cells (AS) were processed in parallel.
(B) Effects of RAD18 overexpression on PCNA modification throughout the cell cycle. Cells were treated and analyzed as in (A). Note that, in this panel, monoubiquitylated PCNA is abundant enough to be detected by the anti-ubiquitin antibody, which recognizes this form very poorly (Hoege et al., 2002). Rad18 was detected in total cell extracts.

Influence of the Type of DNA Damage on PCNA Ubiquitylation
(A) PCNA ubiquitylation after treatment with HU, MMS, NQO, H₂O₂, UV, IR, Bleomycin (Bleo), and CPT at the indicated doses. Modified forms of PCNA were detected as described in the legend to Figure 1.
(B) Sensitivities of WT cells to Bleomycin as monitored by spot assays.
(C) Sensitivities of WT and rad52 cells to CPT.
(D) PCNA ubiquitylation in WT versus rad52 cells. Treatment with damaging agents (50 μg/ml CPT, 0.02% MMS) and detection of the modifications was performed as above.
(E) Induction of the DNA damage checkpoint after treatment with selected DNA-damaging agents. Rad53 phosphorylation, monitored by western blot analysis, was used as an indicator of checkpoint activation.
(F) Quantification of ssDNA after treatment with MMS, HU, or CPT by radioactive random-primed labeling of nondenatured genomic DNA. The graph shows the average labeling efficiencies with standard deviations (in cpm) from triplicate experiments, corrected for the amount of template DNA as determined by real-time PCR.

In Vivo Association of Rad18 with ssDNA
(A) Association of Rad18^9myc with a replication fork in HU-treated cells detected by ChIP after release from G1 arrest. Rad18^9myc was precipitated (anti-myc) from formaldehyde-crosslinked cells at the indicated times, and associated DNA was quantified by real-time PCNA using primers specific for an early-firing origin, ARS607 (black); a sequence 4 kbp removed from ARS607 (gray); and a late-firing origin, ARS501 (white). AS, asynchronous cells. Error bars in all panels represent combined standard deviations from three independent amplifications.
(B) Rfa1^9myc is detectable by ChIP at sequences adjacent to an HO-induced DSB. Enrichment of sequences adjacent to the HO site (MAT) relative to those at an unrelated locus (ACT1) in immunoprecipitates (anti-myc) from crosslinked cells was followed over the indicated time using real-time PCR as in (A). ChIP assays were performed in WT (black) and rad52 mutants (white).
(C) PCNA (Pol30^9myc) is not detectable by ChIP at the site of an HO-induced DSB in WT (black) or rad52 (white) cells.
(D) Association of Rad18^9myc next to a DSB, analyzed as above in WT (black) and rad52 (white) cells.

Active Replication Forks Are Required for PCNA Ubiquitylation
(A) Experimental strategy to prevent the formation of replication forks in the cdc7^ts mutant.
(B) FACS profiles of WT and cdc7^ts cells subjected to the treatment outlined in (A).
(C) Progression through the cell cycle in WT and cdc7^ts cells as monitored by Clb2 and Sic1 levels. Note that at 37°C WT cells have completed mitosis and re-entered G1 phase at 80 min, whereas cdc7^ts mutants accumulate in G2/M with abnormally high Clb2 and low Sic1 levels. The asterisk indicates a band crossreactive to the Sic1 antibody. Detection of phosphoglycerate kinase (PGK) served as a loading control.
(D) Damage-induced PCNA ubiquitylation in WT and cdc7^ts cells after treatment as outlined in (A). S phase samples in cdc7^ts cells and in WT at 24°C were taken 40 min after release, whereas the S phase sample in the WT at 37°C was taken after 25 min due to the faster cell-cycle progression at this temperature (see [B] and [C]).
(E) CDC7 is dispensable for PCNA ubiquitylation. Damage-induced PCNA modification was examined in asynchronous cultures of isogenic WT, bob1, and bob1 cdc7Δ cells.

Effects of DNA on the Interaction between Rad18 and RPA
(A) Recruitment of ^HisVSVRad18 to RPA-coated ssDNA under high-salt conditions. A 75 nt 5′-biotinylated oligonucleotide was immobilized on streptavidin agarose and preincubated with increasing amounts of purified yeast RPA, and retention of ^HisVSVRad18 was analyzed by western blotting. RPA and ^HisVSVRad18 were allowed to bind in a buffer containing 15 mM KCl and 250 mM NaCl.
(B) DNA binding of ^HisVSVRad18 under low-salt conditions. The experiment was performed exactly as in (A), except that the proteins were allowed to bind to the ssDNA at 15 mM KCl.
(C) Preincubation of short oligonucleotides with ^GSTRfa1(182–421) at a ratio of 1:1 or 5:1 enhances interaction with Rad18. Pull-down assays were performed as described in the legend to Figure 5F.
(D) Deletion (Δ) or mutation (^∗) of the Rad18 SAP domain impairs PCNA ubiquitylation in vivo. The rad18 deletion mutant was complemented by the indicated alleles, and PCNA modifications were analyzed as in Figure 1. Presence of the mutant proteins was verified by immunoprecipitation and western blot (lower panel).
(E) The Rad18 SAP domain is required for protection from DNA damage in vivo. Spot assays for NQO and MMS sensitivity were performed on a rad18 deletion mutant complemented by the indicated RAD18 constructs.

RPA Is Required for PCNA Ubiquitylation
(A) PCNA is ubiquitylated normally in the rfa1-t11 and rfa1-t33 mutants. MMS-induced PCNA ubiquitylation was detected as described in the legend to Figure 1 in WT (RFA1) and two rfa1 mutants.
(B) DNA association of the mutant protein encoded by rfa1-t33 is indistinguishable from the WT protein at 25°C and 37°C. Whole-cell extracts (W) were fractionated by centrifugation into soluble (S) and chromatin-associated (C) material. Proteins were detected by western blot. The distributions of the chromatin-associated Orc6 protein and soluble PGK served as controls for the quality of fractionation.
(C) Experimental strategy to deplete Rfa1^td from yeast cells. In the rfa1-td strain, the construct encoding the heat-labile Rfa1^td protein is controlled by a doxycycline (Dox)-repressible promoter, and UBR1, encoding the E3 responsible for degradation of Rfa1^td, is induced by galactose (Gal). Rfa1^td remains stable at 25°C in raffinose (Raf) medium. Temperature effects were excluded by returning the cells to 25°C without allowing resynthesis of Rfa1^td before releasing them into S phase.
(D) DNA replication proceeds after depletion of the Rfa1^td protein. FACS samples were taken before and 50 min after release from G1 arrest following the scheme shown in (C).
(E) Loss of damage-induced PCNA ubiquitylation after depletion of the Rfa1^td protein. ^HisPCNA and its modified forms were detected as in Figure 1. Rfa1 was detected in total cell extracts.
(F) Chromatin association of PCNA in RFA1 and rfa1-td. Chromatin-binding assays were performed as described in (B). Note that the Rfa1^td protein remaining after depletion is entirely associated with chromatin. The degron tag is partially cleaved from the protein during extract preparation.

Interaction of Rad18 with the RPA Complex
(A) Protein-protein interactions between Rad18, Rad5, and the RPA subunits Rfa1, Rfa2, and Rfa3 in the yeast two-hybrid system. The open reading frames were expressed as fusions to the Gal4 activation (AD) and DNA-binding domains (BD), and the presence of the constructs was confirmed by growth on selective medium (-LW). Positive interactions were scored by growth on plates lacking histidine (-HLW) and stronger interactions on plates lacking histidine and adenine (-AHLW). Interactions between Rad18 and Rad5 (Ulrich and Jentsch, 2000) and between the individual RPA subunits are shown as internal controls.
(B) Coomassie-stained gel of recombinant ^HisVSVRad18 isolated in complex with untagged Rad6 from baculovirus-infected insect cells.
(C) Interaction between purified recombinant RPA, covalently coupled to Sepharose, and ^HisVSVRad18. ^HisVSVRad18 retained on the RPA beads was eluted in Laemmli buffer and detected by western blotting. BSA-coupled beads served as negative control.
(D) Two-hybrid analysis of the Rad18 domains interacting with Rfa1 and Rfa2. The scheme on the right indicates the RING domain, a Zinc finger (ZF), a SAP domain, and the region relevant for interaction with Rad6. Positions C48 and C190 are indicated by asterisks in the respective mutants.
(E) Two-hybrid analysis of the Rfa1 domains interacting with Rad18. The scheme on the right indicates the regions relevant for ssDNA binding and holocomplex formation as well as a Zinc finger domain. The position of the rfa1-t11 mutation (Umezu et al., 1998) is indicated by an asterisk.
(F) Interaction of Rad18 with the ssDNA-binding domain of Rfa1 and with Rfa2 in vitro. Purified recombinant ^GSTRfa1(182–421) or Rfa2, immobilized on glutathione Sepharose, was incubated with ^HisVSVRad18. Bound material was eluted and analyzed by western blot to detect Rad18 (upper panel) or by Coomassie staining to detect the GST constructs (lower panel).
(G) In vivo interaction of Rad18 with RPA was analyzed by coimmunoprecipitation of yeast Rad18^6HA and Rfa1^9myc by using the indicated antibodies. Strains expressing untagged RAD18 or RFA1 served as negative controls for precipitation of Rad18^6HA and Rfa1^9myc, respectively. In the anti-Rfa1 precipitation, antibody was omitted as a negative control. Bands of higher molecular weight in anti-HA precipitates represent ubiquitylated Rad18^6HA.
(H) Interaction between hRad18 and hRPA in cultured human cells. Immunoprecipitations (anti-FLAG) were performed from extracts derived from HEK293T cells transiently transfected with an expression construct for ^FLAGhRad18. Cells transfected with the empty vector served as negative control. Extracts were partitioned into detergent-soluble (S) and -insoluble (I) material. Note that ^FLAGhRad18 is monoubiquitylated in the soluble fraction, which includes the cytoplasmic material, but not in the chromatin-associated fraction.