Fig 2: Cellular sensitivities to hydroxyurea (HU) and H2O2 of PC3 rad9A+/+ and rad9A-/- cells expressing WT-Rad9A or mutant Rad9A.(A) Cell viability in response to hydroxyurea (HU). rad9A KO PC3 cells were transfected with vector p3XFLAG-CMV-14 (KO control) or vector containing hRad9A cDNA [(FLAG-WT-hRad9A, KO + WT), (FLAG-DM-Rad9A, KO + DM), (FLAG-LD-Rad9A, KO + LD) or (FLAG-K220A-Rad9A, KO + K220A)]. Plated cells were treated with 2 mM HU for 2 h or left untreated (control). After recovery for 72 hrs, the plates were incubated for 2 h in regular medium containing 40 μg/ml of neutral red and analyzed for cell viability. (B) Cell viability in response to H2O2. The procedures are like (A) except those cells were treated with 80 μM H2O2 for 1 hr. The percentages (%) of control are the ratios of colony numbers of treated cells over those of untreated cells. Cell viability was scored from three experiments. The error bars reported are the standard deviations of the averages and P-value was calculated using ANOVA followed by separate post hocs analysis. ** and *** represent P <0.05 and P <0.01, respectively.

Fig 3: DNA association of Rad9A is required for HU-induced Chk1 phosphorylation.The control rad9A+/+ (+/+) and rad9A-/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S-317) (A), total Chk1 (B), and β-actin (C). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.

Fig 4: A model of the 9-1-1 complex with DNA based on PCNA-DNA structure.(A) Human 9-1-1 (access # 3A1J) [37] is superimposed to PCNA-DNA structure (access # 5L7C) [47]. Rad9A, Hus1, Rad1, and PCNA are colored in green, blue, magenta, and gray, respectively. DNA is shown in gray. (B) PCNA protein structure is omitted from (A). DNA is shown in orange. (C) Superimposed structure of Rad9A, N-terminal domain of Hus1, and DNA. Side chains of R22, K78, S160, K220, and R223 of hRad9A and K93 of hHus1 are shown.

Fig 5: DNA binding activities of hRad9A mutants.Binding with blunt-ended HC40:HG40-DNA was performed by EMSA. The protein:DNA complexes were fractionated on 6% non-denature polyacrylamide gels. (A) DNA binding of wild-type (WT)-hRad9A deletion constructs. (B) DNA binding of Rad9A94-266 constructs containing mutations. DM represents R150A/R223A double mutant. (C) DNA binding of different Rad9A constructs containing mutations. TM, QM1, and LD represent K11A/K15A/R22A, K11A/K15A/R22A/K78A, and loop deletion (Δ160–163) mutants, respectively. Protein concentrations are indicated above each lane. Enzyme-bound and free DNA bands were quantified by ImageQuant Total software (GE Healthcare) to calculate the percentage of DNA binding. Based on the EMSA data, the Rad9A protein concentration resulting in a 50% of band shift was estimated as KD. The KD values and relative DNA binding are indicated below each panel. Free DNA substrate is marked by an arrow and the protein:DNA complexes including shifted discrete bands or smear regions are marked.