Cell-cycle checkpoint genes

Details Cell-cycle checkpoint genes Cell-cycle checkpoint genes

1999-05-11

2003-10-14

Caputa, Anthony C. (Department: 1642)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C435S006120, C435S325000, C435S320100, C435S069100

Reexamination Certificate

active

06632936

ABSTRACT:

The present invention relates to a class of checkpoint genes which control progression through the cell cycle in eukaryotic cells.
BACKGROUND OF THE INVENTION
Control of the cell cycle is fundamental to the growth and maintenance of eukaryotic organisms, from yeasts to mammals. Eukaryotic cells have evolved control pathways, termed “checkpoints” which ensure that individual steps of the cell cycle are completed before the next step occurs. In response to DNA damage, cell survival is increased both by direct DNA repair mechanisms and by delaying progression through the cell cycle. Depending on the position of the cell within the cycle at the time of irradiation, DNA damage in mammalian cells can prevent (a) passage from G1 into S phase, (b) progression through S phase or (c) passage from G2 into mitosis. Such checkpoints are thought to prevent deleterious events such as replication of damaged DNA and the segregation of fragmented chromosomes during mitosis (Hartwell and Kastan, 1994).
The rad3 gene of
Schizosaccharomyces pombe
is required for the checkpoints that respond to DNA damage and replication blocks. Rad3 is a member of the lipid kinase subclass of kinases which possess regions having sequence homology to the lipid kinase domain of the p110 subunit of phosphatidylinositol-3 kinase (PI-3 kinase). This subclass also includes the ATM protein defective in ataxia-telangiectasia patients. Cells from aeaxia telangiectasia patients (AT cells) have lost the delay to S phase following irradiation and are said to display radio resistant DNA synthesis (Painter and Young, 1989). AT cells irradiated in S phase accumulate in G2 with lethal damage, presumably as a consequence of attempting to replicate damaged DNA. AT cells irradiated during G2 display a different phenotype, they do not arrest mitosis after DNA damage, and progress through mitosis with damaged DNA (Beamish and Lavin, 1994). Mutations at the A-T locus, to which the ATM gene has been mapped, thus result in disruption of several checkpoints required for an appropriate response to ionising radiation. Other members of this lipid kinase subclass include: Tel1p (Greenwell et al. 1985). a gene involved in maintaining proper telomere length in
Saccharomyces cerevisiae;
Esr1p; Mec1p and the product of the
Drosophila melanogaster mei
-41 checkpoint gene (Hari et al. 1995).
DISCLOSURE OF THE INVENTION
We have analyzed the
S. pombe
rad3 gene and found that it has a full length amino acid sequence of 2386 amino acids, not the 1070 amino acids described by Seaton et al. 1992. We have determined that this is the direct homologue of
S. cerevisiae
Esr1p, and that it shares the same overall structure as the ATM gene. The C-terminal region of the rad3 protein contains a lipid kinase domain, which is required for Rad3 function We have shown that Rad3 is capable of self association. We have also identified a protein kinase activity associated with Rad3.
Further, we have found a human homologue to rad3. This gene, which we have named ATR (ataxia and rad related), displays significantly higher homology to rad3 than it does to the ATM gene.
The human ATR cDNA sequence is set out as Seq. ID No. 1. The amino acid sequence of the ORF from nucleotides 80 and 8011 is set out as Seq. ID No. 2.
The DNA sequence of the open reading frame (ORF) of rad3 is shown as Seq. ID. No. 3. The 2386 amino acid translation of the gene (nucleotides 585 to 7742 of Seq. ID No. 3) is shown as Seq. ID. No. 4.
Accordingly, in a first aspect, the invention provides the ATR protein of Seq. ID. 2 and homologues thereof, polypeptide fragments thereof, as well as antibodies capable of binding the ATR protein or polypeptide fragments thereof. ATR proteins, homologues and fragments thereof are referred to below as polypeptides of the invention.
In another aspect, the present invention provides a polynucleotide in substantially isolated form capable of hybridising selectively to Seq.ID No. 1 or to the complement (i.e. opposite stand) thereof. Also provided are polynucleotides encoding polypeptides of the invention.
Such polynucleotides will be referred to as a polynucleotide of die invention. A polynucleotides of the invention includes DNA of Seq.ID Nos. 1 and fragments thereof capable of selectively hybridising to this gene.
In a further aspect, the invention provides recombinant vectors carrying a polynucleotide of the invention, including expression vectors, and methods of growing such vectors in a suitable host cell, for example under conditions in which expression of a protein or polypeptide encoded by a sequence of the invention occurs.
In an additional aspect, the invention provides kits comprising polynucleotides, polypeptides or antibodies of the invention and methods of using such kits in diagnosing the presence of absence of ATR and its homologues, or variants thereof, including deleterious ATR mutants.
The invention further provides assay methods for screening candidate substances for use as compounds for inhibiting or activating ATR activity, or the activity of mutated forms of ATR which are deficient in checkpoint activity. The invention also provides assay methods for screening candidate substances for use as compounds for inhibiting interactions between ATR and other compounds that interact with ATR, including ATR itself.
In a related aspect, the invention also provides a polynucleotide sequence of Seq. ID No. 3 in substantially isolated form, and the protein of Seq. ID No. 4 in substantially isolated form, and novel fragments and variants thereof.
DETAILED DESCRIPTION OF THE INVENTION
A. Polynucleotides
Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the an. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention.
Polynucleotides of the invention capable of selectively hybridizing to the DNA of Seq. ID No. 1 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the corresponding DNA of Seq. ID No. 1 over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
Any combination of the above mentioned degrees of homology and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 80% homologous over 25, preferably 30 nucleotides forms one aspect of the invention, as does a polynucleotide which is at least 90% homologous over 40 nucleotides.
Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any mean

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