DNA damage induce gamma-tubulin-RAD51 nuclear complexes in mammalian cells.
Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, gamma-tubulin. This association was confirmed by the reverse immunoprecipitation with gamma-tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and gamma-tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and gamma-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.
Lesca
Claire
C
IPBS, UMR 5089, CNRS Université Paul Sabatier, 205 Route de Narbonne, 31077 Toulouse Cedex 4, France.
Germanier
Maryse
M
Raynaud-Messina
Brigitte
B
Pichereaux
Carole
C
Etievant
Chantal
C
Emond
Stéphane
S
Burlet-Schiltz
Odile
O
Monsarrat
Bernard
B
Wright
Michel
M
Defais
Martine
M
D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D046912:Multiprotein Complexes / Q000378:metabolism; D051135:Rad51 Recombinase; D016196:S Phase / Q000502:physiology; D014404:Tubulin / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D014404:Tubulin; C495153:RAD51 protein, human; D051135:Rad51 Recombinase