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bioRxiv · 10.1101/070276

Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament

Maureen M Mundia·Alissa C Magwood·Mark D Baker
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Abstract

In this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3 polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3 polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3 polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3 polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

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Maureen M Mundia, Alissa C Magwood, Mark D Baker. 2016-08-18. Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament. https://doi.org/10.1101/070276

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