Journal
PLOS ONE
Volume 5, Issue 12, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0015073
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Funding
- National Institutes of Health [P01-DK58327, PO1 HL59412, PO1 HL51811]
- Juvenile Diabetes Research Foundation (JDRF)
- Alpha One Foundation
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Background: DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive. Methodology/Principal Findings: In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins. Conclusion/Significance: Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.
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