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Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth.

ABSTRACT: Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here, we show that Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homolog Uls1 accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, nondamage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci, and Rdh54 predominates for removal of nondamage-associated foci.

SUBMITTER: Shah PP

PROVIDER: S-EPMC2946244 | biostudies-other | 2010 Sep

REPOSITORIES: biostudies-other

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Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth.

Shah Parisha P PP Zheng Xiuzhong X Epshtein Anastasiya A Carey Jeffrey N JN Bishop Douglas K DK Klein Hannah L HL

Molecular cell 20100901 6

Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here, we show that Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, ...[more]

PMID: 20864034

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Project description:BackgroundPreviously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.MethodologyUsing fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase ADomain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.SignificanceThe use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

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Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth.

Publications

Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth.

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