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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.003632

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Processing of recombinant AAV genomes occurs in specific nuclear structures that overlap with foci of DNA-damage-response proteins

Tiziana Cervelli^1,*, Jose Alejandro Palacios^2,*, Lorena Zentilin², Miguel Mano², Rachel A. Schwartz, Matthew D. Weitzman³ and Mauro Giacca^1,2,

¹ Molecular Biology Laboratory, Scuola Normale Superiore, AREA della Ricerca del CNR, Via Moruzzi 1, Pisa, Italy
² Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, Italy
³ Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

Author for correspondence (e-mail: giacca{at}icgeb.org)

Accepted 30 October 2007

Despite increasing utilization of rAAV vectors in gene transfer applications, several aspects of the biology of these vectors remain poorly understood. We have visualized the conversion of rAAV vector genomes from single-stranded to double-stranded DNA in real time. We report that rAAV DNA accumulates into discrete foci inside the nucleus. These rAAV foci are defined in number, increase in size over time after transduction, are relatively immobile, and their presence correlates with the efficiency of cell transduction. These structures overlap with, or lie in close proximity to, the foci in which proteins of the MRN (MRE11-RAD50-NBS1) complex as well as the MDC1 protein accumulate after DNA damage. The downregulation of MRN or MDC1 by RNA interference markedly increases both the formation of rAAV foci and the extent of rAAV transduction. Chromatin immunoprecipitation experiments indicate that the MRE11 protein associates with the incoming rAAV genomes and that this association decreases upon cell treatment with DNA damaging agents. These findings are consistent with a model whereby cellular DNA-damage-response proteins restrict rAAV transduction by negatively regulating rAAV genome processing.

Key words: AAV, DNA repair, MRN complex, Viral vectors

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