ATR-2 cell cycle checkpoint

Details ATR-2 cell cycle checkpoint ATR-2 cell cycle checkpoint

2001-09-21

2004-11-09

Nickol, Gary B. (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C536S023200

Reexamination Certificate

active

06815532

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel polynucleotides encoding cell cycle checkpoint polypeptides.
BACKGROUND
The mitotic cell cycle is the process by which a cell creates an exact copy of its chromosomes and then segregates each copy into two cells. The sequence of events of the cell cycle is carefully regulated such that cell division does not occur until the cell has completed DNA replication and, DNA replication does not occur until cells have completed mitosis. If a cell is exposed to DNA damage, the damage is repaired before the cell undergoes cell division. Regulation of these processes ensures that an exact copy of DNA is propagated to the daughter cells. The cell cycle has been divided into four phases: G1, S, G2, and M. During the G1 phase, cells undergo activities that prepare for DNA replication. S, or synthesis, phase begins as cells initiate DNA replication and ends with the formation of two identical copies of each chromosome. G2, the stage that begins after replication is complete, is when cells ensure that they contain components needed for mitosis. M phase, or mitosis, is the stage at which the cells divide each identical chromosome into two daughter cells.
Cells have mechanisms for sensing correct cell cycle progression and exposure to DNA damage, and proteins involved in these sensing mechanisms are termed checkpoints. Checkpoints signal cell cycle arrest to allow for completion of relevant events or repair of DNA damage. There are checkpoints that monitor progression through the cycle at G1, S, G2, and M. DNA damage checkpoints also exist at these stages of the cell cycle. Failure to correct DNA damage may signal the cell to undergo programmed cell death or apoptosis.
Members of the phosphatidylinositol kinase (PIK)-related family of kinases are involved in cell cycle checkpoints and DNA damage repair. To date, five PIK-related protein kinases have been identified. Genes in this family, which includes ATM, ATR, FRAP and DNA-PKcs, encode large proteins (280-450 kD) that exhibit homology to kinases at the carboxy terminus. While the predicted amino acid sequences of the kinase domains are most closely related to lipid kinases, all have been shown to function as protein kinases, and, presumably, each of these proteins participate in a signal transduction cascade leading to cell cycle arrest, cell cycle progression, and/or DNA repair.
The ataxia-telangiectasia mutated (ATM) gene product has been shown to play a role in a DNA damage checkpoint in response to ionizing radiation (IR). Patients lacking functional ATM develop the disease ataxia-telangiectasia (A-T). Symptoms of A-T include extreme sensitivity to irradiation, cerebellar degeneration, oculocutaneous telangiectasias, gonadal deficiencies, immunodeficiencies, and increased risk of cancer [Lehman and Carr,
Trends in Genet.
11:375-377 (1995)]. Fibroblasts derived from these patients show defects in G1, S, and G2 checkpoints [Painter and Young,
Proc. Natl. Acad. Sci. (USA)
77:7315-7317 (1980)] and are defective in their response to irradiation. ATM is thought to sense double strand DNA damage caused by irradiation and radiomimetic drugs, and to signal cell cycle arrest so that the damage can be repaired.
The DNA-stimulated protein kinase, DNA-PKcs has been demonstrated to play an important role in repair of double strand breaks. Mice defective in DNA-PK demonstrate immunodeficiencies and sensitivity to irradiation. In addition, these mice are defective in V(D)J recombination. These results suggest that DNA-PK plays a role in repairing normal double strand DNA breaks generated during V(D)J recombination, as well as double strand breaks generated by DNA damaging agents. While DNA-PK defective cells have not been shown to be deficient in cell cycle checkpoints, it is reasonable to assume that the cell cycle must arrest, if only transiently, in order to repair double strand breaks.
ATR has been found to act as a checkpoint protein stimulated by agents that cause double strand DNA breaks, agents that cause single strand DNA breaks, and agents that block DNA replication [Cliby, et al.,
EMBO J.
17:159-169 (1998); Wright, et al.,
Proc. Natl. Acad. Sc. (USA)
95:7445-7450 (1998)]. Overexpression of ATR in muscle cells on iso-chromosome 3q results in a block to differentiation, gives rise to abnormal centrosome numbers and chromosome instability, and abolishes the G1 arrest in response to irradiation [Smith, et al.
Nat. Genetics
19:39-46 (1998)]. Overexpression of a dominant negative mutant of ATR sensitizes cells to irradiation and cisplatinum [Cliby, et al., supra] and the cells fail to arrest in G2 in response to irradiation. ATR is found associated with chromosomes in meiotic cells where DNA breaks and abnormal DNA structures that persist as a result of the process of meiotic recombination [Keegan, et al,
Genes Dev.
10:2423-2437 (1996)]. These data suggest that ATR, like ATM, senses DNA damage and effects a cell cycle arrest in order to allow for DNA repair.
FRAP, the target of the potent immunosuppressent rapamycin, has been demonstrated to be involved in the control of translation initiation and progression through the G1 phase of the cell cycle in response to nutrients [Kuruvilla and Shrieber,
Chemistry and Biology
6:R129-R136 (1999)]. FRAP regulates translation initiation by phosphorylation of the p70
S6K
protein kinase and the 4E-BP1 translation regulator. While ATM, ATR, and DNA-PK are thought to sense lesions in nucleic acids, FRAP is thought to sense intracellular levels of amino acids pools. In cells lacking proper nutrients that are amino acid starved, uncharged amino acid levels rise. FRAP may sense these uncharged amino acids, become activated, and signal G1 cell cycle arrest [Kuruvilla and Shreiber, supra].
In yeast, Tor1p and Tor2p proteins show significant homology to FRAP. Both Tor1p and Tor2p are sensitive to rapamycin and both are involved in initiation of translation as well as G1 progression in response to nutrient conditions. Tor2p also plays a role in organization of actin cytoskeleton, but this activity is not blocked by rapamycin. These observations suggest that Tor2p stimulates two distinct signal transduction pathways.
An additional PIK-related family member, TRRAP, was recently identified as a member of a protein complex containing the cell cycle regulators, c-myc and E2F-1 [McMahon et al.,
Cell
94:363-374 (1998)]. While TRRAP shows significant sequence homology to the protein kinase domain of the other PIK-related kinases, the protein lacks critical residues required for protein kinase activity. Studies have failed to show protein kinase activity, but others have shown that TRRAP contains a histone acetyltransferase (HAT) activity. Interestingly, overexpression of TRRAP dominant inhibiting mutants or anti-sense constructs of TRRAP blocked oncogenic transformation of cultured cells transformed by c-myc or the viral oncogene, E1A [McMahon et al., supra]. These results suggest that TRRAP also plays an important role in regulating cell cycle progression and preventing oncogenesis.
In general, the proteins in this family of kinases play important roles in surveillance of DNA and cell cycle progression in order to insure genetic integrity from generation to generation. All cancer cells have a dysfunctional cell cycle and continue through the cell cycle in an inappropriate manner, either by failing to respond to negative growth signals or by failing to die in response to the appropriate signal. In addition, most cancer cells lack genomic integrity and often have an increased chromosome count compared to normal cells. Inhibitors of cell cycle checkpoints or DNA damage repair in combination with the cytotoxic agents may force cancer cells to die by forcing them to continue to progress through the cell cycle in the presence of DNA damaging agents such that they undergo catastrophic events that lead to cell death. Further, inhibitors of cell cycle progress

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