Methods and compositions utilizing rad51

Inactive Publication Date: 2003-07-31
HAAF THOMAS +4
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  • Abstract
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  • Claims
  • Application Information

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Benefits of technology

0015] In an additional aspect, the invention provides methods for screening for a bioactive agent capable of modulating the activity of Rad51. The method comprises the steps of adding a candidate bioactive agent to a sample of Rad51, and determining an alteration in the biological activity of Rad51. The method may also comprise adding a candidate bioactive agent to a

Problems solved by technology

Moreover attempts to generate homozygous rad51-/-embryonic stem cells have not been successful.
However, although the MN assay is a convenient in situ method to monitor cytogenetic effects, the understanding of the connection between initial DNA damage and formation of MN is still poor.
Loss of wild-type p53 function renders cells resistan

Method used

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  • Methods and compositions utilizing rad51
  • Methods and compositions utilizing rad51
  • Methods and compositions utilizing rad51

Examples

Experimental program
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Effect test

example 1

Immunofluorescent Staining of Human Breast Cancer Cells

[0092] Breast tumour cells have mutated p53 and have various types of chromosomal aberrations like insertions, deletions, rearrangements, amplifications etc. Recombination proteins such as Rad51 could evidently participate in such processes. In order to better understand the role of uncontrolled recombination and its role in tumour formation and progression, the status of Rad51 protein in breast tumour cells by staining them with anti Rad51 antibodies was done.

[0093] Detailed methods of cloning and expression of HsRad51 gene in E. coli, purification of recombinant HsRad51 protein with six histidine residues at it's aminoterminal end and preparation of ployclonal antibodies against HsRad51 protein were described previously by Haaf, Golub et al. 1995, supra, which is expressly incorporated herein by reference.

[0094] Immunofluorescent staining with anti-Rad51 protein antibodies. Monolayer cultures of different cell substrates (see ...

example 2

Nuclear Foci of Human Recombination Protein Rad51 in Nucleotide Excision Repair Defective Cells

[0104] Eurkaryotic cells have several different mechanisms for repairing damaged DNA (for review see R. Wood, 1996). One of the major pathway is nucleotide excision repair (NER), which excises damage within oligomers that are 25-32 nucleotides long. Patients with recessive heredity disorder XP have defects in one of several enzymes, which participate in ER. There are seven XP groups (XP-A to XP-G), which have defects in the initial steps of the DNA excision repair.

[0105] DNA damage is removed several-fold faster from transcribed genes than from non-transcribed, mainly due to preferential NER of the transcribed strand (for review see Hanawalt, 1994). This mechanism does not function in Cockayne's syndrome (CS) patients.

[0106] NER defective cells, evidently, sustain increased amount of DNA damage. Thus we evaluated NER defective cells from XP and CS cells for an increased amount of Rad51 pro...

example 3

Higher Order Nuclear Structures of Rad51 and Its Exclusion Into Micronuclei After Cell Damage

[0116] Previous studies have revealed a time- and dose-dependent increase of nuclear HsRad51 protein foci after DNA damage introduced into the genome by various agents (Haaf et al., 1995, supra). Here we show that when the damaged cells are allowed to recover, these Rad51 foci form specific higher-order nuclear structures. Finally, all the focally concentrated Rad51 protein is eliminated into micronuclei that undergo apoptotic genome fragmentation. Treatment of cells with clastogens and aneuploidogens implements a mechanism that affects the nuclear distribution of Rad51 protein and targets Rad51 foci, most likely along with irreversibly damaged DNA into micronuclei. To examine the role of Rad51 protein in DNA repair and cell proliferation, we have analyzed the intranuclear distribution of overexpressed Rad51 protein during the cell cycle and in cell populations proceeding through apoptosis.

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Abstract

In accordance with the objects outlined above, the present invention provides methods of diagnosing individuals at risk for a disease state which results in aberrant Rad51 loci. The methods comprise determining the distribution of Rad51 foci in a first tissue type of a first individual, and then comparing the distribution to the distribution of Rad51 foci from a second normal tissue type from the first individual or a second unaffected individual. A difference in the distributions indicates that the first individual is at risk for a disease state which results in aberrant Rad51 loci. Preferred disease states include cancer and disease states associated with apoptosis.

Description

[0001] This is a continuing application of 60 / 035,834, filed Jan. 30, 1997 and 60 / 045,668, filed May 6, 1997, both of which are expressly incorporated by reference herein.FIELD OF THE INVENTION[0002] The invention relates to methods of diagnosis and screening utilizing Rad51 molecules.BACKGROUND OF THE INVENTION[0003] Homologous recombination is a fundamental process which is important for creating genetic diversity and for maintaining genome integrity. In E. coli RecA protein plays a central role in homologous genetic recombination in vivo and promotes homologous pairing of double-stranded DNA with single-stranded DNA or partially single-stranded DNA molecules in vitro. Radding, C. M. (1988). Homologous pairing and strand exchange promoted by Escherichia coli RecA protein. Genetic Recombination. Washington, American Society for Microbiology. 193-230; Radding, C. M. (1991). J. Biol. Chem. 266: 5355-5358; Kowalczykowski, et al., (1994). Annu. Rev. Biochem. 63: 991-1043. In the yeast ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18G01N33/15G01N33/50G01N33/574
CPCG01N33/5011G01N33/5743G01N33/57415G01N33/574
Inventor HAAF, THOMASGOLUB, EFIM ILYAREDDY, GURUCHARANRADDING, CHARLES MEYERWARD, DAVID C.
Owner HAAF THOMAS
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