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: Structure of the 9-1-1 complex.(A) Top view of the human 9-1-1 complex. The crystal structure 91−272-1-1 is shown (RCSB codes:3A1J) [37]. The extreme C-terminal domain of Rad9A (residues 267–391) is not shown because its structure has not been determined. The key differences among the three subunits are circled in red. The DNA in the inner channel of the 9-1-1 ring is bound by Rad9A and the N-terminal domain of Hus1 [43] shown in orange and blue arrows, respectively. (B) Basic residues and S160 of hRad9A as well as R18 and K93 of hHus1 in the channel of the 9-1-1 ring are labeled. A small loop (residues 160–163, with sequence SPAL) of hRad9A projected into the inner ring surface is circled in red. (C) A side view of hRad9A structure looking from inside of the ring. Residues 1–93 are colored dark violet and residues 94–266 are colored green. A small loop (residues 160–163) is circled in red.

Fig 4: 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 5: 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.