RAD9A

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.[6] This complex plays a role in DNA base excision repair. Hus1 binds and stimulates MYH DNA glycosylase which stimulates base excision repair.[7] 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,[8] 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.[7]

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.[8] 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.[8] 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.[8] 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.

Rad9 is activated by multiple phosphorylations by cyclin dependent kinases and activates Rad53 through Mec1 downstream.[9] Mrc1 has also been shown to work cooperatively to recruit Rad53 to damaged DNA.[10] 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.[11] Rad9 has also been shown to interact with p53 and can even mimic certain functions of p53.[5]

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.[5]

RAD9A has been shown to interact with:

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.[26] 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.[27] 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.[27]