Human checkpoint kinase, HCDS1, compositions and methods

Details Human checkpoint kinase, HCDS1, compositions and methods Human checkpoint kinase, HCDS1, compositions and methods

2000-04-07

2002-07-02

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S183000, C435S069100, C530S387100, C530S350000, C536S023100

Reexamination Certificate

active

06413755

ABSTRACT:

BACKGROUND OF THE INVENTION
The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of checkpoint controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2, M) which correlate with distinct cell morphology and biochemical activity, and cells withdrawn from the cell-cycle are said to be in G0, or non-cycling state. When cells within the cell-cycle are actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin,
GENES VI
(Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of organization that result in the formation of chromosomes. Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin,
GENES VI
(Oxford University Press, Oxford, GB, Chapter 5, 1997).
Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989
, Science
, 246: 629-634). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997
, Science
, 274: 1664-1671). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et at., 1997
, Mol. Cell Biol
., 8: 1-11; Jin et al., 1996
, J. Cell Biol
., 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997
, Cancer Res
., 57: 5168-5178).
Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the G1-S transition (Hoffmann et al., 1994
, EMBO J
., 13: 4302-4310; Jinno et al., 1994
, EMBO J
., 13: 1549-1556), whereas Cdc25B and Cdc25C are required for the G2-M transition (Gabrielli et al., 1996
, J. Cell Sci
., 7: 1081-1093; Galaktionov et al., 1991
, Cell
, 67: 1181-1194; Millar et al., 1991
, Proc. Natl. Acad. Sci. USA
, 88: 10500-10504; Nishijima et al., 1997
, J. Cell Biol
., 138: 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known.
Much of our current knowledge about checkpoint control has been obtained from studies using budding (
Saccharomyces cerevisiae
) and fission (
Schizosaccharomyces pombe
) yeast. A number of reviews of our current understanding of cell cycle checkpoints in yeast and higher eukaryotes have recently been published (Hartwell & Kastan, 1994
, Science
, 266: 1821-1828; Murray, 1994
, Current Biology
, 6: 872-876; Elledge, 1996
, Science
, 274: 1664-1672; Kaufmann & Paules, 1996
, FASEB J
., 10: 238-247). In the fission yeast six gene products, rad1
+
, rad3
+
, rad9
+
, rad17
+
, rad26
+
, and hus1
+
have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cds1
+
has been identified as being required for the DNA-replication dependent checkpoint and rad27
+
/chk1
+
has been identified as required for the DNA-damage dependent checkpoint in yeast.
Several of these genes have structural homologues in the budding yeast and further conservation across eukaryotes has recently been suggested with the cloning of two human homologues of
S. pombe
rad3
+
: ATM (ataxia telangiectasia mutated) (Savitsky et al., 1995
, Science
, 268: 1749-1753) and ATR (ataxia telangiectasia and rad3
+
related)(Bentley et al, 1996
, EMBO J
., 15: 6641-6651; Cimprich et al., 1996
, Proc. Natl. Acad. Sci. USA
, 93: 2850-2855) and of a human homologue of
S. pombe
rad9
+
(Lieberman et al., 1996
, Proc. Natl. Acad. Sci. USA
, 93: 13890-13885).
While much is known about yeast checkpoint proteins and genes, this knowledge is not fully predictive of the existence of corresponding human genes or proteins, or their effector role in human cell-cycle control and regulation.
In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of cancer, it is important to identify and characterize human checkpoint proteins and to identify mediators of their activity.
SUMMARY OF THE INVENTION
The present invention is directed to the discovery of a novel human checkpoint kinase gene hCDS1, protein and constructs and methods for the production and use of HCDS1.
In particular, the present invention encompasses a nucleic acid sequence which encodes for hCDS1, consisting of the nucleic acid sequence of SEQ ID NO.: 1. In particular, the invention encompasses the nucleic acid sequence from position 66 to 1694 of the nucleic acid sequence of SEQ ID NO.: 1, which translates into the hCDS1 protein. The present invention also encompasses nucleic acid constructs, vectors, plasmids, cosmids and the like which contain the nucleic acid sequence of SEQ ID NO.: 1. In particular, the present invention provides for nucleic acid vector constructs which contain the nucleic acid sequence of SEQ ID NO.: 1 and are capable of expressing protein from this nucleic acid sequence. The present invention encompasses nucleic acid vectors that are suitable for the transformation of host cells, whether eukaryotic or prokaryotic, suitable for incorporation into viral vectors, or suitable for in vitro protein expression. The present invention further embodies the nucleic acid sequence of SEQ ID NO.: 1 in tandem with, or otherwise in conjunction with additional nucleic acids for the generation of fusion protein products containing at least the functional segment of the protein encoded for by the nucleic acid of SEQ ID NO.: 1. The present invention also encompasses the nucleic acid of SEQ ID NO.: 1 adapted for use as a naked DNA transformant for incorporation and expression in target cells. The present invention also provides for anti-sense DNA molecule formulations which are the complement to the nucleic acid sequence of SEQ ID NO.: 1, and fragments thereof, whether complementary to contiguous or discontinuous portions of the nucleic acid sequence of SEQ ID NO.: 1. The present invention also provides for compositions incorporating modified nucleotides or backbone components which encode for the nucleic acid sequence of SEQ ID NO.: 1, its complement, or fragments thereof. Such modified nucleotides and nucleic acids are known in the art (see for example Verma et al.,
Ann. Rev. Biochem
. 67: 99-134 (1998)). Thus the present invention encompasses modified nucleic acids which incorporate, for example, intemucleotide linkage modification, base modifications, sugar modification, nonradioactive labels, nucleic acid cross-linking, and altered backbones including PNAs (polypeptide nucleic acids).
The present invention provides for the novel human checkpoint kinase protein hCDS1, which consists of the amino acid sequence of SEQ ID NO.: 2. The invention encompasses hCDS1 protein produced by recombinant DNA technology and expressed in vivo or in vitro. The invention thus encompasses hCDS1 protein produced by transformed host cells in small-scale or large-scale production. The invention encompasses complete hCDS1 protein, in either glycosylated or unglycosylated forms, produced by either eukaryotic or prokaryotic cells. The present invention provides for hCDS1 protein expressed from mammalian, insect, plant, bacterial, fungal, or any other suitable host cell. The present invention encompasses hCDS1 protein that is produced as a fusion protein product, conjugated to a solid support, or hDCS1 protein which is labeled with any chemical, radioactive, fluorescent, chemiluminescent or otherwise detectable marker. The present invention also provides for hDCS1 protein isolated from natural sources and enriched in pu

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