ABSTRACT
Recentstudies have shown that wild-type and recombinant adeno-associated
virus (AAV and rAAV) genomes persist in human tissue predominantly as
double-stranded (ds) circular episomes derived from input linear
single-stranded virion DNA. Using self-complementary recombinant AAV
(scAAV) vectors, we generated intermediates that directly transition to
ds circular episomes. The scAAV genome ends are palindromic
hairpin-structured terminal repeats, resembling a double-stranded break
repair intermediate. Utilizing this substrate, we found cellular DNA
recombination and repair factors to be essential for generating
circular episomal products. To identify the specific cellular proteins
involved, the scAAV circularization-dependent vector was used as a
reporter in 19 mammalian DNA repair-deficient cell lines. The results
show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of
the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient
scAAV genome circularization. We further demonstrated that the scAAV
genome requires ATM and DNA-PKCS, but not NBS1, to
efficiently convert to a circular form in nondividing cells in vivo
using transgenic mice. These studies identify specific pathways
involved for further elucidating viral and cellular mechanisms of DNA
maintenance important to the viral life cycle and vector
utilizations.
Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes