Methods and compositions in checkpoint signaling
AssigneeBaylor College of Medicine
InventorsStephen J. Elledge, David K. Cortez, Lee Zou
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Abstract
The present invention is directed to compositions and methods regarding the signaling for the presence of DNA damage or replication stress and activating cell cycle checkpoints. Specifically, ATRIP was identified as an interactor with ATR, a member of the phosphatidylinositol kinase-related protein family that includes ATM and DNA-PK. In some embodiments, the present invention is directed to ATRIP and ATR acting as mutually dependent partners in cell cycle checkpoint signaling pathways.
Claims74
As a composition of matter, an isolated nucleic acid sequence comprising SEQ ID NO:
- 1.
As a composition of matter, a purified polypeptide sequence comprising SEQ ID NO:
- 2.
As a composition of matter, an isolated genomic DNA sequence encoding the polypeptide comprising SEQ ID NO:
- 2.
A vector comprising the nucleic acid sequence of SEQ ID NO:
- 1, wherein the expression of the nucleic acid sequence is regulated by nucleic acid sequences operatively linked to said nucleic acid sequence comprising SEQ ID NO:
- 1.
As a composition of matter, a fusion polypeptide comprising at least a portion of an ATRIP polypeptide and a non-ATRIP polypeptide.
A method of preventing or delaying a cell from entering into mitosis following damage to DNA in said cell, comprising contacting a cell with an ATRIP polypeptide in an amount effective to prevent or delay entry of said cell into mitosis.
A method of preventing or delaying proliferation of a cell, said cell comprising a defective ATRIP polynucleotide or polypeptide, comprising contacting the cell with ATRIP in an amount effective to prevent or delay said cell proliferation.
A method for identifying an agent that interacts with an ATRIP polypeptide, comprising the steps of:
- providing said ATRIP polypeptide;
- providing a candidate agent to said ATRIP polypeptide;
- and
- assaying for an interaction between said candidate agent and said ATRIP polypeptide.
A method for identifying a modulator of ATRIP activity comprising:
- (a) providing a candidate modulator;
- (b) admixing the candidate modulator with an isolated compound, cell, or suitable experimental animal comprising ATRIP;
- (c) measuring one or more characteristics of the compound, cell or animal in step (b);
- and
- (d) comparing the characteristic measured in step (c) with the characteristic of the compound, cell or animal in the absence of said candidate modulator,
- wherein a difference between the measured characteristics indicates that said candidate modulator is a modulator of the compound, cell or animal comprising ATRIP.
A method of screening for an agent that interacts with ATR, comprising the steps of:
- introducing into a cell
- a first nucleic acid expressing a fused test peptide/DNA binding domain;
- and
- a second nucleic acid expressing a fused ATR polypeptide/DNA activation domain;
- and
- assaying for an interaction between said test peptide and said ATR polypeptide by measuring binding between said DNA binding domain and said DNA activation domain, wherein said interaction between said test peptide and said ATR polypeptide indicates said test peptide is said agent.
A method of screening for an agent that interacts with ATRIP, comprising the steps of:
- introducing into a cell
- a first nucleic acid expressing a fused test peptide/DNA binding domain;
- and
- a second nucleic acid expressing a fused ATRIP polypeptide/DNA activation domain;
- and
- assaying for an interaction between said test peptide and said ATRIP polypeptide by measuring binding between said DNA binding domain and said DNA activation domain, wherein said interaction between said test peptide and said ATRIP polypeptide indicates said test peptide is said agent.
A method of screening in vitro for an active compound for the treatment of cancer, comprising the steps of:
- obtaining a cell, wherein said cell comprises a nucleic acid sequence having a reporter sequence and wherein the expression of said reporter sequence is controlled by an ATRIP regulatory nucleic acid sequence;
- exposing a test agent to said cell;
- and
- measuring a change in said expression, wherein said change indicates said test agent is said active compound.
A method of screening in vitro for an active compound for the treatment of cancer, comprising the steps of:
- obtaining a cell, wherein said cell includes a nucleic acid sequence comprising a reporter sequence and wherein the expression of said reporter sequence is controlled by an ATR regulatory nucleic acid sequence;
- exposing a test agent to said cell;
- and
- measuring a change in said expression, wherein said change indicates said test agent is said active compound.
A method for detecting an ATRIP polypeptide in a sample, comprising:
- obtaining the sample suspected of containing the ATRIP polypeptide;
- providing an antibody that recognizes an ATRIP polypeptide;
- mixing the sample and the antibody under conditions wherein the antibody can bind to the ATRIP protein;
- and
- detecting the binding.
A method for detecting a cancer cell, comprising:
- obtaining a sample comprising a cell having an ATRIP polynucleotide;
- and
- identifying a defect in said ATRIP polynucleotide.
A method for detecting a cancer cell, comprising:
- obtaining a sample comprising a cell having an ATRIP polypeptide;
- and
- identifying a defect in said ATRIP polypeptide.
A method for treating a cancer cell having a defective ATRIP polynucleotide or polypeptide, comprising administering to said cell a non-defective ATRIP polynucleotide or polypeptide.
A monoclonal antibody that binds immunologically to a polypeptide comprising SEQ ID NO:
- 2, or an antigenic fragment thereof.
A polyclonal antisera, antibodies of which bind immunologically to a polypeptide comprising SEQ ID NO:
- 2, or an antigenic fragment thereof.
A method of identifying an agent that interacts with ATR, comprising:
- providing a cell that is reduced for ATR function;
- providing to said cell an ATR polypeptide;
- providing to said cell a candidate agent;
- and
- assaying for an interaction between said ATR polypeptide and said candidate agent.
A method of identifying an agent that interacts with ATRIP, comprising:
- providing a cell that is reduced for ATR or ATRIP function;
- providing to said cell an ATRIP polypeptide;
- providing to said cell a candidate agent;
- and
- assaying for an interaction between said ATRIP polypeptide and said candidate agent.
A method for identifying an agent that interacts with an ATRIP polypeptide, comprising the steps of:
- providing said ATRIP polypeptide;
- providing a candidate agent to said ATRIP polypeptide;
- and
- assaying for an interaction between said candidate agent and said ATRIP polypeptide.
A method of identifying an agent that inhibits binding of ATRIP to a ssDNA molecule, comprising the steps of:
- providing a ssDNA molecule;
- providing an ATRIP polypeptide;
- introducing to said ssDNA molecule a test agent;
- and
- assaying for an interaction between said ATRIP polypeptide and said ssDNA molecule, wherein when said interaction does not occur, said test agent is said agent that inhibits binding.
A method of identifying an agent that inhibits a function of an ATR/ATRIP complex, comprising the steps of:
- providing a ssDNA molecule;
- providing a replication protein A (RPA) polypeptide, wherein said ssDNA molecule and said RPA polypeptide form a ssDNA/RPA complex;
- providing an ATR/ATRIP-phosphorylatable protein, wherein said ATR/ATRIP-phosphorylatable protein binds to said ssDNA or said ssDNA/RPA complex;
- providing an ATRIP polypeptide;
- providing an ATR polypeptide, wherein said ATRIP polypeptide and said ATR polypeptide form an ATR/ATRIP complex;
- providing a test agent;
- and
- assaying phosphorylation status of said ATR/ATRIP-phosphorylatable protein, wherein when said ATR/ATRIP-phosphorylatable protein is not phosphorylated following said providing of said ATRIP polypeptide, said test agent is identified as said agent that inhibits function of said ATR/ATRIP complex.
A method of identifying an agent that promotes cell death, comprising the steps of:
- providing a ssDNA/replication protein A (RPA) complex;
- providing an ATRIP polypeptide;
- introducing to said ssDNA/RPA complex a test agent;
- and
- assaying for an interaction between said ATRIP polypeptide and said ssDNA/RPA complex, wherein when said interaction does not occur, said test agent is said agent that promotes cell death.
A method of identifying an agent that promotes cell death, comprising the steps of:
- providing a ssDNA molecule;
- providing a replication protein A (RPA) polypeptide, wherein said ssDNA molecule and said RPA polypeptide form a ssDNA/RPA complex;
- providing an ATR/ATRIP-phosphorylatable protein, wherein said ATR/ATRIP-phosphorylatable protein binds to said ssDNA or said ssDNA/RPA complex;
- providing an ATRIP polypeptide;
- providing an ATR polypeptide, wherein said ATRIP polypeptide and said ATR polypeptide form an ATR/ATRIP complex;
- providing a test agent;
- and
- assaying phosphorylation status of said ATR/ATRIP-phosphorylatable protein, wherein when said ATR/ATRIP-phosphorylatable protein is not phosphorylated following said providing of said ATRIP polypeptide, said test agent is identified as said agent that promotes cell death.
Dependencies are inferred from the claim text.