DNA damage induce γ-tubulin–RAD51 nuclear complexes in mammalian cells

Abstract

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, c-tubulin. This association was confirmed by the reverse immunoprecipitation with c-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 c-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 c-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.

Mitotic crisis: The unmasking of a novel role for RPA

“…47 A direct involvement of RAD51 and paralogues with the centrosome is not yet established but is suggested by the finding that RAD51 can physically interact with γ-tubulin, a centrosome-associated factor. 49 As a second example, while the recombination mediator BRCA2 is predominantly a nuclear factor, a fraction of the BRCA2 pool localizes to centrosomes in the S and early M phases. A reduction in the level of cytoplasmic and hence centrosomal BRCA2 causes centrosome amplification and binucleation of cells.…”

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells

“…This cell cycle arresting effect clearly suggests that nuclear accumulation of β-tubulin, which does not occur under normal circumstances, is a process of pathophysiological significance and may represent a defense mechanism against stress or malignant transformation. In that respect, it is interesting to note that GFPtagged α-tubulin accumulates reversibly in nuclei of cultured tobacco cells, in response to low temperature (Schwarzerova et al, 2006), whereas nuclear β-and γ-tubulin have been so far detected primarily in cancer or transformed cells (Lesca et al, 2005;WalssBass et al, 2002;Xu and Luduena, 2002;Yeh and Luduena, 2004;.…”

Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase

“…We found that 4.1R depletion delays transitioning into S phase in untransformed RPE-1 cells, supporting the important role of 4.1R in centrosome structure. Although 4.1R depletion does (1,9,34,45,55). In addition to its functions at interphase centrosomes and in cell cycle regulation, 4.1R is required for formation of mitotic bipolar spindles and for proper cytokinesis.…”