DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex

Abstract

The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction. The N-terminal globular domain (residues 1-133) had 3.7-fold better DNA binding affinity than the C-terminal globular domain (residues 134-266) of Rad9A1-266. Rad9A1-266 binds DNA 16-, 60-, and 30-fold better than Rad9A1-133, Rad9A134-266, and Rad9A94-266, respectively, indicating that different regions cooperatively contribute to DNA binding. We show that basic residues including K11, K15, R22, K78, K220, and R223 are important for DNA binding. The reductions on DNA binding of Ala substituted mutants of these basic residues show synergistic effect and are dependent on their residential Rad9A deletion constructs. Interestingly, deletion of a loop (residues 160-163) of Rad9A94-266 weakens DNA binding activity by 4.1-fold as compared to wild-type (WT) Rad9A94-266. Cellular sensitivity to genotoxin of rad9A knockout cells is restored by expressing WT-Rad9Afull. However, rad9A knockout cells expressing Rad9A mutants defective in DNA binding are more sensitive to H2O2 as compared to cells expressing WT-Rad9Afull. Only the rad9A knockout cells expressing loop-deleted Rad9A mutant are more sensitive to hydroxyurea than cells expressing WT-Rad9A. In addition, Rad9A-DNA interaction is required for DNA damage signaling activation. Our results indicate that DNA association by Rad9A is critical for maintaining cell viability and checkpoint activation under stress.

Fig 1. 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 2. The hRad9A constructs and mutants used in DNA binding assay are depicted.

Numbers represent residues of hRad9A. Residues 1–93 are colored dark violet and residues 94–266 are colored green except residues 125–140 (colored pink) which are the interdomain connecting loop (IDCL) between the N- and C-terminal globular domains. The C-terminal tail (C-tail) (colored blue) of hRad9A is not required for DNA binding.

Fig 3. Purified His-tagged hRad9A constructs.

Proteins were analyzed by 12% -15% SDS-polyacrylamide gel electrophoresis and stained by Coomassie Blue. Corresponding protein bands are indicated by arrows.

Fig 4. 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 Rad9A^94-266 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 K_D. The K_D 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.

Fig 5. 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 6. 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 7. 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.