PT  - JOURNAL ARTICLE
AU  - Maureen M. Mundia
AU  - Alissa C. Magwood
AU  - Mark D. Baker
TI  - Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament <em>in vivo</em>
AID  - 10.1101/070276
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 070276
4099  - http://biorxiv.org/content/early/2016/08/18/070276.short
4100  - http://biorxiv.org/content/early/2016/08/18/070276.full
AB  - 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.