REC2 kinase

Details REC2 kinase REC2 kinase

1998-09-21

2001-01-16

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S194000

Reexamination Certificate

active

06174694

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns the field of molecular genetics and medicine. Particularly, it concerns a gene encoding a protein that is a kinase and is involved in cell cycle regulation and the repair of damaged genomic DNA in mammalian cells. The gene and protein, termed herein, respectively hsREC2 and hsRec2, is in the same supergene family as the mammalian protein having homologous pairing and strand transfer activities, RAD51 and was isolated because of its homology to the homologous pairing and strand transfer protein of
Ustilago maydis.
Due to this relationship the same gene and protein is termed elsewhere RAD51B and Rad51B.
BACKGROUND OF THE INVENTION
2.1 The Structure and Function of hsREC2
During the life of every organism the DNA of its cells is constantly subjected to chemical and physical events that cause alterations in its structure, i.e., potential mutations. These potential mutations are recognized by DNA repair enzymes found in the cell because of the mismatch between the strands of the DNA. To prevent the deleterious effects that would occur if these potential mutations became fixed, all organisms have a variety of mechanisms to repair DNA mismatches. In addition, higher animals have evolved mechanisms whereby cells having highly damaged DNA, undergo a process of programmed death (“apoptosis”).
The association between defects in the DNA mismatch repair and apoptosis inducing pathways and the development, progression and response to treatment of oncologic disease is widely recognized, if incompletely understood, by medical scientists. Chung, D. C. & Rustgi, A. K., 1995, Gastroenterology 109:1685-99; Lowe, S. W., et al., 1994, Science 266:807-10. Therefore, there is a continuing need to identify and clone the genes that encode proteins involved in DNA repair and DNA mismatch monitoring.
Studies with bacteria, fungi and yeast have identified three genetically defined groups of genes involved in mismatch repair processes. The groups are termed, respectively, the excision repair group, the error prone repair group and the recombination repair group. Mutants in a gene of each group result in a characteristic phenotype. Mutants in the recombination repair group in yeast result in a phenotype having extreme sensitivity to ionizing radiation, a sporulation deficiency, and decreased or absent mitotic recombination. Petes, T. D., et al., 1991, in Broach, J. R., et al., eds., The Molecular Biology of the Yeast Saccharomyces, pp. 407-522 (Cold Spring Harbor Press, 1991).
Several phylogenetically related genes have been identified in the recombination repair group: recA, in
E. Coli,
Radding, C. M., 1989, Biochim. Biophys. Acta 1008:131-145; RAD51 in
S. cerevisiae,
Shinohara, A., 1992, Cell 69:457-470, Aboussekhra, A. R., et al., 1992, Mol. Cell. Biol. 12:3224-3234, Basile, G., et al., 1992, Mol. Cell. Biol. 12:3235-3246; RAD57 in
S. cerevisiae,
Gene 105:139-140; REC2 in
U. maydis,
Bauchwitz, R., & Holloman, W. K., 1990, Gene 96:285-288, Rubin, B. P., et al., 1994, Mol. Cell. Biol. 14:6287-6296. A third
S. cerevisiae
gene DMC1, is related to recA, although mutants of DMC1 show defects in cell-cycle progression, recombination and meiosis, but not in recombination repair.
The phenotype of REC2 defective
U. maydis
mutants is characterized by extreme sensitivity to ionizing radiation, defective mitotic recombination and interplasmid recombination, and an inability to complete meiosis. Holliday, R., 1967, Mutational Research 4:275-288. UmREC2, the REC2 gene product of
U. maydis,
has been extensively studied. It is a 781 amino acid ATPase that, in the presence of ATP, catalyzes the pairing of homologous DNA strands in a wide variety of circumstances, e.g., UmREC2 catalyzes the formation of duplex DNA from denatured strands, strand exchange between duplex and single stranded homologous DNA and the formation of a nuclease resistant complex between identical strands. Kmiec, E. B., et al., 1994, Mol. Cell. Biol. 14:7163-7172. UmREC2 is unique in that it is the only eukaryotic ATPase that forms homolog pairs, an activity it shares with the
E. coli
enzyme recA.
U.S. patent application Ser. No. 08/373,134, filed Jan. 17, 1995, by W. K. Holloman and E. B. Kmiec discloses REC2 from
U. maydis,
methods of producing recombinant UmREC2 and methods of its use. Prior to the date of the present invention a fragment of human REC2 cDNA was available from the IMAGE consortium, Lawrence Livermore National Laboratories, as plasmid p153195. Approximately 400 bp of the sequence of p153195 had been made publicly available on dbEST database.
The scientific publication entitled: Isolation of Human and Mouse Genes Based on Homology to REC2, July 1997, Proc. Natl. Acad. Sci. 94, 7417-7422 by Michael C. Rice et al., discloses the sequences of murine and human Rec2, of the human REC2 cDNA, and discloses that irradiation increases the level of hsREC2 transcripts in primary human foreskin fibroblasts. The scientific publication Albala et al., December 1997, Genomics 46, 476-479 also discloses the sequence of the same protein and cDNA which it terms RAD51 B. A sequence that is identical to hsREC2 except for the C-terminal 14 nucleotides of the coding sequence and the 3′-untranslated sequence was published by Cartwright R., et al., 1998, Nucleic Acids Research 26, 1653-1659 and termed hsR51h2. It is believed that hsREC2 and hsR51h2 represent alternative processing of the same primary transcript. The parent application of this application was published as WO 98/11214 on Mar. 19,1998.
The structure of hsREC2 is also disclosed in application Ser. No. 60/025,929, filed Sep. 11, 1996, application Ser. No. 08/927,165, filed Sep. 11, 1997, and patent publication WO 98/11214, published Mar. 19, 1998.
2.2 Cell Cycle Regulation
The eukaryotic cell cycle consists of four stages, G
1
, S (synthesis), G
2
, and M (mitosis). The underlying biochemical events that determine the stage of the cell and the rate of progression to the next stage is a series of kinases, e.g., cdk2, cdc2, which are regulated and activated by labile proteins that bind them, termed cyclins, e.g., cyclin D, cyclin E, Cyclin A . The activated complex in turn phosphorylates other proteins which activates the enzymes that are appropriate for each given stage of the cycle. Reviewed, Morgan, D. O., 1997, Ann. Rev. Cell. Dev. Biol. 15, 261-291; Clurman, B. E., & Roberts, J. M., 1998, in The Genetic Basis of Human Cancer, pp. 173-191 (ed. by Vogelstein, B., & Kinzer K. W., McGraw Hill, N.Y.) (hereafter Vogelstein)
The cell cycle contains a check point in G
1
. Under certain conditions, e.g., chromosomal damage or mitogen deprivation, a normal cell will not progress beyond the check point. Rb and p53 are proteins involved in the G
1
check point related to mitogen deprivation and chromosomal damage, respectively. Inactivating mutations in either of these proteins results, in concert with other mutations, in a growth transformed, i.e., malignant, cell. The introduction of a copy of the normal p53 or Rb gene suppresses the transformed phenotype. Accordingly genes, such as p53 or Rb, whose absence is associated with transformation are termed “tumor suppressor” genes. A frequent cause of familial neoplastic syndromes is the inheritance of a defective copy of a tumor suppressor gene. Reviewed Fearson, E. R., in
Vogelstein
pp. 229-236.
The level of p53 increases in response to chromosomal damage, however, the mechanism which mediates this response is unknown. It is known that p53 can be phosphorylated by a variety of kinases and that such phosphorylation may stabilize the p53 protein. Reviewed Agarwal, M. L., et al., Jan. 2, 1998, J. Biol. Chem. 273, 1-4.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected discovery that hsRec2 is a serine kinase that phosphorylates several proteins that control the cell cycle, particularly cyclin E and p53. The invention permits the phosphorylation of the cell cycle control proteins at sites that are physiologically elevant. In addition, the discovery of the e

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