Cell cycle checkpoint control protein RAD9A is a protein that in humans is encoded by the RAD9Agene.[1]Rad9 has been shown to induce G2 arrest in the cell cycle in response to DNA damage in yeast cells. Rad9 was originally found in budding yeast cells but a human homolog has also been found and studies have suggested that the molecular mechanisms of the S and G2 checkpoints are conserved in eukaryotes.[2] Thus, what is found in yeast cells are likely to be similar in human cells.
This gene product is highly similar to S. pombe rad9, a cell cycle checkpoint protein required for cell cycle arrest and DNA damage repair in response to DNA damage. This protein is found to possess 3' to 5' exonuclease activity, which may contribute to its role in sensing and repairing DNA damage. It forms a checkpoint protein complex with Rad1 and Hus1. This is also known as the Rad9-Rad1-Hus1 or 9-1-1 complex. This complex is recruited by checkpoint protein Rad17 to the sites of DNA damage, which is thought to be important for triggering the checkpoint-signaling cascade. Use of alternative polyA sites has been noted for this gene.[3] This complex plays a role in DNA base excision repair. Hus1 binds and stimulates MYH DNA glycosylase which stimulates base excision repair.[4] Rad9 binds with the strongest affinity to DNA which attaches the complex to damaged DNA. Rad1 recruits other base excision factors. Previous research has suggested that Rad9 is not necessary to repair DNA,[5] but it does not mean it can still play a role in DNA damage repair. If Rad9 is mutated there may be other pathways or mechanisms in DNA repair that can compensate for a loss of function.[4]
History
Rad9 was first found as a gene that promotes G2 cell cycle arrest in response to DNA damage in Saccharomyces cerevisiae by Weinert et al.[5] The group irradiated yeast cells to induce DNA damage and tested many different mutants. They tested 7 rad mutants and all of the mutants underwent G2 arrest as normal, except for one, the rad9 mutant. The rad9 mutant did not undergo G2 arrest and instead proceeded through the cell cycle and many of the cells died because the DNA was never repaired.[5] From this they suspected that Rad9 is necessary to invoke G2 cell cycle arrest. To confirm this they tested a double mutant of rad9 with DNA repair deficient-strain rad52 and found that the cell failed to arrest in G2 further proving that a functioning Rad9 gene is needed to induce G2 arrest. They then used MBC, a microtubule inhibitor, to synthetically arrest the cell in G2 in order to test if the Rad9 gene was necessary to also repair DNA. The found that when the rad9 mutant was arrested in G2, irradiated to induce DNA damage, and left arrested in G2 by MBC for 4 hours, the cell was able to repair DNA and divide normally.[5] This result suggested that Rad9 is not necessary to repair DNA. They concluded that Rad9 is an important gene that is crucial to arrest the cell in G2 and ensures fidelity of chromosome transmission but is not necessary to repair DNA.
Interactions
Rad9 is activated by multiple phosphorylations by cyclin dependent kinases and activates Rad53 through Mec1 downstream.[6] Mrc1 has also been shown to work cooperatively to recruit Rad53 to damaged DNA.[7] After the 9-1-1 complex Rad9 is extensively phosphorylated by Mec1 which can trigger self-association of more Rad9 oligomers on the chromosomes. Further phosphorylation generates binding sites for Rad53 which also gets activated by Mec1 to pursue its target in the cell cycle control system. Rad9 doesn’t do the DNA repair itself, it is just an adaptor protein that sends the signal.[8]
Rad9 has also been shown to interact with p53 and can even mimic certain functions of p53.[2]
Rad9 has been shown to be able to bind to the same promoter region as p53 that transactivates p21, which halts progression of the cell cycle by inhibiting cyclins and CDK’s. In addition to transactivating p21, Rad9 can also regulate transcription of the base excision repair gene NEIL by binding p53-like response elements in the gene promoter.[2]
The Rad9 protein contains a carboxy-terminal tandem repeat of the BRCT (BRCA1 carboxyl terminus) motif, which is found in many proteins involved in DNA damage repair.[23] This motif is necessary for Rad9 to function. When the BRCT motif was removed, cell survival severely decreased compared to wild type Rad9. Rad9 is normally hyperphosphorylated after DNA damage.[24] and the rad9 mutants without the BRCT motif displayed no phosphorylation so it is possible that the phosphorylation sites are located on this domain. The same mutant was also not able to phosphorylate Rad53 downstream.[24]
↑ 5.05.15.25.3Weinert TA, Hartwell LH (July 1988). "The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae". Science. 241 (4863): 317–22. doi:10.1126/science.3291120. PMID3291120.
↑Bacal J, Moriel-Carretero M, Pardo B, Barthe A, Sharma S, Chabes A, Lengronne A, Pasero P (November 2018). "Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage". The EMBO Journal. 37 (21). doi:10.15252/embj.201899319. PMID30158111.
↑Morgan DO (2012). The Cell Cycle: Principles of Control. Oxford: Oxford University Press.
↑Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, et al. (October 2005). "Towards a proteome-scale map of the human protein-protein interaction network". Nature. 437 (7062): 1173–8. doi:10.1038/nature04209. PMID16189514.
↑ 12.012.1Komatsu K, Miyashita T, Hang H, Hopkins KM, Zheng W, Cuddeback S, Yamada M, Lieberman HB, Wang HG (January 2000). "Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis". Nat. Cell Biol. 2 (1): 1–6. doi:10.1038/71316. PMID10620799.
↑Xiang SL, Kumano T, Iwasaki SI, Sun X, Yoshioka K, Yamamoto KC (October 2001). "The J domain of Tpr2 regulates its interaction with the proapoptotic and cell-cycle checkpoint protein, Rad9". Biochem. Biophys. Res. Commun. 287 (4): 932–40. doi:10.1006/bbrc.2001.5685. PMID11573955.
↑Cai RL, Yan-Neale Y, Cueto MA, Xu H, Cohen D (September 2000). "HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins". J. Biol. Chem. 275 (36): 27909–16. doi:10.1074/jbc.M000168200. PMID10846170.
↑ 15.015.115.2Dufault VM, Oestreich AJ, Vroman BT, Karnitz LM (Dec 2003). "Identification and characterization of RAD9B, a paralog of the RAD9 checkpoint gene". Genomics. 82 (6): 644–51. doi:10.1016/s0888-7543(03)00200-3. PMID14611806.
↑ 16.016.1Volkmer E, Karnitz LM (January 1999). "Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex". J. Biol. Chem. 274 (2): 567–70. doi:10.1074/jbc.274.2.567. PMID9872989.
↑ 17.017.1Griffith JD, Lindsey-Boltz LA, Sancar A (May 2002). "Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy". J. Biol. Chem. 277 (18): 15233–6. doi:10.1074/jbc.C200129200. PMID11907025.
↑ 18.018.1Hirai I, Wang HG (July 2002). "A role of the C-terminal region of human Rad9 (hRad9) in nuclear transport of the hRad9 checkpoint complex". J. Biol. Chem. 277 (28): 25722–7. doi:10.1074/jbc.M203079200. PMID11994305.
↑Rauen M, Burtelow MA, Dufault VM, Karnitz LM (September 2000). "The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9". J. Biol. Chem. 275 (38): 29767–71. doi:10.1074/jbc.M005782200. PMID10884395.
↑Mäkiniemi M, Hillukkala T, Tuusa J, Reini K, Vaara M, Huang D, Pospiech H, Majuri I, Westerling T, Mäkelä TP, Syväoja JE (August 2001). "BRCT domain-containing protein TopBP1 functions in DNA replication and damage response". J. Biol. Chem. 276 (32): 30399–406. doi:10.1074/jbc.M102245200. PMID11395493.
↑Soulier J, Lowndes NF (May 1999). "The BRCT domain of the S. cerevisiae checkpoint protein Rad9 mediates a Rad9-Rad9 interaction after DNA damage". Current Biology. 9 (10): 551–4. doi:10.1016/S0960-9822(99)80242-5. PMID10339432.
↑ 24.024.1Sun Z, Hsiao J, Fay DS, Stern DF (July 1998). "Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint". Science. 281 (5374): 272–4. doi:10.1126/science.281.5374.272. PMID9657725.
Further reading
Lieberman HB (2006). "Rad9, an evolutionarily conserved gene with multiple functions for preserving genomic integrity". J. Cell. Biochem. 97 (4): 690–7. doi:10.1002/jcb.20759. PMID16365875.
Volkmer E, Karnitz LM (1999). "Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex". J. Biol. Chem. 274 (2): 567–70. doi:10.1074/jbc.274.2.567. PMID9872989.
Komatsu K, Miyashita T, Hang H, Hopkins KM, Zheng W, Cuddeback S, Yamada M, Lieberman HB, Wang HG (2000). "Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis". Nat. Cell Biol. 2 (1): 1–6. doi:10.1038/71316. PMID10620799.
Bessho T, Sancar A (2000). "Human DNA damage checkpoint protein hRAD9 is a 3' to 5' exonuclease". J. Biol. Chem. 275 (11): 7451–4. doi:10.1074/jbc.275.11.7451. PMID10713044.
Hang H, Lieberman HB (2000). "Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression". Genomics. 65 (1): 24–33. doi:10.1006/geno.2000.6142. PMID10777662.
Cai RL, Yan-Neale Y, Cueto MA, Xu H, Cohen D (2000). "HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins". J. Biol. Chem. 275 (36): 27909–16. doi:10.1074/jbc.M000168200. PMID10846170.
Burtelow MA, Kaufmann SH, Karnitz LM (2000). "Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage". J. Biol. Chem. 275 (34): 26343–8. doi:10.1074/jbc.M001244200. PMID10852904.
Rauen M, Burtelow MA, Dufault VM, Karnitz LM (2000). "The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9". J. Biol. Chem. 275 (38): 29767–71. doi:10.1074/jbc.M005782200. PMID10884395.
Komatsu K, Wharton W, Hang H, Wu C, Singh S, Lieberman HB, Pledger WJ, Wang HG (2000). "PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition". Oncogene. 19 (46): 5291–7. doi:10.1038/sj.onc.1203901. PMID11077446.
Chen MJ, Lin YT, Lieberman HB, Chen G, Lee EY (2001). "ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation". J. Biol. Chem. 276 (19): 16580–6. doi:10.1074/jbc.M008871200. PMID11278446.
Burtelow MA, Roos-Mattjus PM, Rauen M, Babendure JR, Karnitz LM (2001). "Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex". J. Biol. Chem. 276 (28): 25903–9. doi:10.1074/jbc.M102946200. PMID11340080.
Mäkiniemi M, Hillukkala T, Tuusa J, Reini K, Vaara M, Huang D, Pospiech H, Majuri I, Westerling T, Mäkelä TP, Syväoja JE (2001). "BRCT domain-containing protein TopBP1 functions in DNA replication and damage response". J. Biol. Chem. 276 (32): 30399–406. doi:10.1074/jbc.M102245200. PMID11395493.
St Onge RP, Besley BD, Park M, Casselman R, Davey S (2001). "DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein". J. Biol. Chem. 276 (45): 41898–905. doi:10.1074/jbc.M105152200. PMID11551919.
Xiang SL, Kumano T, Iwasaki SI, Sun X, Yoshioka K, Yamamoto KC (2001). "The J domain of Tpr2 regulates its interaction with the proapoptotic and cell-cycle checkpoint protein, Rad9". Biochem. Biophys. Res. Commun. 287 (4): 932–40. doi:10.1006/bbrc.2001.5685. PMID11573955.
Griffith JD, Lindsey-Boltz LA, Sancar A (2002). "Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy". J. Biol. Chem. 277 (18): 15233–6. doi:10.1074/jbc.C200129200. PMID11907025.
Hang H, Zhang Y, Dunbrack RL, Wang C, Lieberman HB (2002). "Identification and characterization of a paralog of human cell cycle checkpoint gene HUS1". Genomics. 79 (4): 487–92. doi:10.1006/geno.2002.6737. PMID11944979.