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Involvement of cellular double-stranded DNA break binding proteins in processing of the recombinant adeno-associated
L Zentilin1, A Marcello, M Giacca
1Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy.
Abstract:
Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.
Insights
Recombinant adeno-associated virus (rAAV) transduction is poor in cultured cells but influenced by DNA repair. Rad52 protein binding to the rAAV genome may promote its processing via homologous recombination.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Recombinant adeno-associated virus (rAAV) exhibits poor transduction efficiency in cultured cells compared to in vivo postmitotic tissues.
- rAAV transduction is enhanced by DNA-damaging agents and elevated in cells with defects in ataxia telangiectasia mutated (ATM), indicating genomic instability involvement.
- DNA double-stranded break (DSB) repair pathways are implicated in regulating rAAV transduction efficiency.
Purpose of the Study:
- To investigate the role of DNA double-stranded break (DSB) repair pathways in modulating recombinant adeno-associated virus (rAAV) transduction efficiency.
- To identify specific DNA repair proteins that interact with the rAAV genome during transduction.
Main Methods:
- Quantitative chromatin immunoprecipitation was employed to detect protein-viral DNA interactions within transduced cells.
- Analysis of rAAV transduction efficiency in cells with genetic defects in key DNA repair proteins, specifically Ku86 and Rad52.
Main Results:
- Ku86 and Rad52 proteins were found to associate with the viral DNA of rAAV within transduced cells.
- rAAV transduction efficiency was significantly increased in Ku86-defective cells.
- Conversely, rAAV transduction was inhibited in Rad52 knockout cells, suggesting a critical role for Rad52.
Conclusions:
- DNA double-stranded break (DSB) repair pathways, particularly involving Ku86 and Rad52, play a regulatory role in rAAV transduction.
- The findings suggest that Rad52 protein binding to the rAAV genome is involved in processing the vector genome, potentially through a homologous recombination pathway.