Disease relevance of RAD9

• In yeast, TDP mutation confers a 1000-fold hypersensitivity to camptothecin in the presence of an additional mutation of RAD9 gene [Pouliot, J.J., Yao, K.C., Robertson, C.A. & Nash, H.A. (1999) Science 286, 552-555] [1].
• It is demonstrated that the pronounced G1 arrest observed in yeast after hyperthermia treatment or exposure to paraquat-generated superoxide radicals does not depend on a functional RAD9 gene [2].
• In the present study, semiquantitative reverse transcription-PCR showed that Rad9 mRNA levels were up-regulated in 52.1% (25 of 48) of breast tumors, and this up-regulation correlated with tumor size (P = 0.037) and local recurrence (P = 0.033) [3].

High impact information on RAD9

• Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points [4].
• This adaptation-defective phenotype was entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest [5].
• In wild-type cells, elimination of a telomere caused a RAD9-mediated cell cycle arrest, indicating that telomeres help cells to distinguish intact chromosomes from damaged DNA [6].
• Curiously, the mitosis-specific checkpoint gene RAD9 is not required for meiotic arrest of dmc1 mutants [7].
• In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage [8].

Biological context of RAD9

• RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae [9].
• In budding yeast, DNA damage can activate a checkpoint surveillance system controlled by the RAD9, RAD53, and MEC1 genes, resulting in a delay in cell cycle progression [10].
• The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage [11].
• We have shown previously that RAD9 also controls the transcriptional induction of a DNA damage regulon (DDR) [12].
• Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase [13].

Anatomical context of RAD9

• RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1 Delta cells [14].
• However, efficient DNA repair can occur in irradiated rad9 cells if irradiated cells are blocked for several hours in G2 by treatment with a microtubule poison [15].
• Rates of survival and checkpoint delay of the mutants after ultraviolet (UV) irradiation were essentially equivalent to those of rad9Delta (null) cells, demonstrating that the BRCT domain is required for Rad9 function [16].
• Treatment of MCF-7 cell line with 5'-aza-2'-deoxycytidine reduced Rad9 mRNA expression and also increased binding of Sp3 to the demethylated intron 2 region [3].

Associations of RAD9 with chemical compounds

• We propose that selenite treatment leads to DNA damage inducing the RAD9-dependent cell cycle arrest [17].
• Interruption of the RAD9 gene, which is involved in DNA-damage-induced cell cycle arrest, had no affect on cisplatin cytotoxicity [18].
• In addition, rad9 rad18 is no more sensitive to MMS than the rad18 single mutant, suggesting that rad9 plays a role within the PRR pathway [19].
• Epistatic analysis showed that rad9 is synergistic to both mms2 and rev3 with respect to killing by methyl methanesulfonate (MMS), and the triple mutant is nearly as sensitive as the rad18 single mutant [19].
• These results indicate that the lysine-dependent function of histone H4 is required for the maintenance of genome integrity, and that DNA damage resulting from the loss of this function activates the RAD9-dependent G2/M checkpoint pathway [20].

Physical interactions of RAD9

• Since the phosphorylated form of Rad9p appears capable of interacting stably with Rad53p in vivo, this phosphorylation response likely controls checkpoint signaling by Rad9p [10].

Enzymatic interactions of RAD9

• Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo [21].
• Thus, Rad53 integrates DNA damage signals by coupling with phosphorylated Rad9 [22].

Regulatory relationships of RAD9

• Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways [23].
• Saccharomyces cerevisiae RAD53 (CHK2) and CHK1 control two parallel branches of the RAD9-mediated pathway for DNA damage-induced G(2) arrest [24].
• Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control [25].
• Loss of Stn1 function activated the RAD9 and MEC3 G2/M checkpoints, therefore confirming that DNA damage is generated [26].
• A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint [27].

Other interactions of RAD9

• Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage [28].
• These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal [9].
• MEC1-dependent phosphorylation of Rad9p in response to DNA damage [10].
• The transcriptional response to DNA damage revealed unexpected and specific antagonism between RAD9 and RAD24 [12].
• In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes [11].

Analytical, diagnostic and therapeutic context of RAD9

• We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains [29].
• Northern blot analysis demonstrated that RAD9 controls the DNA damage-specific induction of a large 'regulon' of repair, replication and recombination genes [30].
• Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage [31].
• Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis [31].
• Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM [32].

References