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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

View larger version (58K): [in this window] [in a new window]   Fig. 1. Visualization of the nuclear sites of ss to ds AAVLacO.14 conversion (rAAV foci) in live GFP-LacR cells. (A) AAVLacO.14 contains the AAV ITRs flanking 14 copies of a sequence corresponding to an 8-mer of the Lac operator sequence cassette (LacO), for a total of 112 LacR binding sites. Inside the transduced cells, the GFP-LacR protein only binds the vector genome after its conversion to dsDNA. (B) Selected images from a time-lapse series of an individual MRC/GFP-LacR cell treated with HU and observed from 3 to 28 hours post infection (p.i.). The location of rAAV foci was monitored through a z-series of images. Along the z axis, images of nuclei were captured at 0.25 µm intervals, and the final images were obtained by projection of the individual sections. A movie showing the formation of these foci in real time is presented in the supplementary material Movie 1. (C) Formation of rAAV foci in MRC/GFP-LacR cells transduced with AAVLacO.14 but not with AAV-LacZ. Representative images taken 24 hours after transduction with the same amounts of vector are shown. Cells were either untreated (nt) or treated with hydroxyurea (HU), camptothecin (campto) or bleomycin (bleo), in conditions known to increase the efficiency of rAAV transduction (Zentilin et al., 2001) (see also Materials and Methods).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Kinetics of rAAV foci formation and nuclear accumulation of rAAV genomes. (A,B) MRC5/GFP-LacR were transduced with AAVLacO.14 either without or after treatment with HU. Live cells were analyzed at different times p.i. by counting the number of cells displaying rAAV foci (A) and the number of foci per cell (B). The mean ± s.d. of 30-60 cells per time point are shown. (C) Cells were transduced with AAV-LacZ either without or after treatment with HU. Nuclear fractions were isolated at different times p.i. and the amount of rAAV genomes in nuclear fractions was determined by real-time quantitative PCR. The mean ± s.d. per time point is shown. Purity of nuclear fractions was confirmed by western blot analysis (see supplementary material Fig. S1).

View larger version (79K): [in this window] [in a new window]   Fig. 3. Colocalization of rAAV foci with DNA-damage-response proteins. (A-E) MRC/GFP-LacR or HeLa/GFP-LacR (where indicated) cells were transduced with AAVLacO.14 and, after 24 hours, fixed and immunostained with anti-NBS1 (Nbs1; A), P-S343-NBS1 (B), MRE11 (Mre11; C), RAD50 (Rad50; D) and MDC1 (Mdc1; E) antibodies. HU and campto: cells that were treated with hydroxyurea (1 mM overnight) or camptothecin (1 nM for 6 hours) prior to AAVLacO.14 transduction. The dotted rectangles indicate the portions of the merged figures enlarged in the rightmost panels. Representative cells of at least 50 analyzed per condition are shown. (F) Number of rAAV foci colocalizing with MRN and MDC1 foci. Quantifications were performed for cells treated with HU (NBS1, P-S343-NBS1, MRE11 and RAD50) or camptothecin (MDC1) as in A-E.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Chromatin immunoprecipitation (ChIP) detects MRE11 (Mre11) bound to rAAV genomes. (A) Cross-linked chromatin at 8, 16 and 24 hours after infection with AAV-LacZ was sonicated and immunoprecipitated with an anti-USF antibody. Co-immunoprecipitated DNA was quantified by real-time PCR using primer pairs and probes in the cellular lamin B2 locus, B48 and B13, and rAAV DNA. B48 is located within the lamin B2 origin of DNA replication, which also encompasses the promoter region of the mitochondrial inner membrane translocase 13 (TIMM13) gene that contains a USF binding site; B13 is 5 kb away from the lamin B2 origin, in a region not containing any gene (Todorovic et al., 2005). The results of these quantifications are expressed as fold enrichment over B13, after normalization for the total amount of input chromatin, as already described (Lusic et al., 2003). The results show that transcription factor USF bound the B48 region, but not B13 or rAAV DNA, irrespective of HU treatment, at 8, 16 and 24 hours p.i. (B) Same as in A but using an antibody against MRE11. The protein was found to specifically bind rAAV DNA, especially at 16 and 24 hours after infection; this interaction was significantly decreased in cells pre-treated with HU.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Silencing of NBS1 increases formation of rAAV foci. (A) Western blotting showing the levels of NBS1 in HeLa cells after treatment with siRNAs against NBS1 (siNbs1) or luciferase (siLuc) at 60 hours after siRNA transfection (corresponding to 24 hours p.i.). The western blotting against tubulin was used as a loading control. (B) Representative images of cells treated with anti-NBS1 or anti-Luc siRNAs followed by transduction with AAV-LacO.14. The images were taken at 24 hours p.i. without HU treatment. (C) Percentage of HeLa cells with detectable AAVLacO.14 foci after silencing of NBS1. Cells were evaluated for the presence of rAAV foci 16 and 24 hours after transduction, in the presence or absence of HU treatment, as indicated. (D) Number of AAVLacO.14 foci per cell after silencing of NBS1. Conditions were the same as in C.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Silencing of NBS1 or MDC1 increases rAAV transduction. (A) β-galactosidase activity measured as relative light units (RLU) in lysates from MRC5 cells after treatment with siRNAs against NBS1 (siNbs1) or luciferase (siLuc), followed by transduction with AAV-LacZ at three multiplicities of infection [m.o.i.; 10000, 5000 and 2500 viral genome particles (vgp) per cell]. Cells were either not treated or were treated overnight with 5 mM HU. (B) Same as in A but using HeLa cells. (C) Efficiency of AAV-LacZ (10,000 vgp/cell) transduction (measured as above) of NBS1-ILB1 cells (mutated in both copies of the NBS1 gene) and NBS1-ILB1/NBS1 cells (in which NBS1 function had been complemented by transfection of full length NBS1). (D) Same as in panel A but using AT5 cells, without HU treatment. (E) Flow cytometry analysis of retrovirally transduced HeLa cells expressing shRNAs against MDC1 (pSR-Mdc1) or β-gal (pSR-LacZ) after infection with AAV-GFP. The upper panel shows the levels of MDC1 protein in the two cell lines. (F) Flow cytometry analysis of HeLa cells transfected with siRNAs against MDC1 (siMdc1) or luciferase. The upper panel shows the levels of MDC1 protein 60 hours after siRNA transfection (24 hours after AAV-GFP transduction).

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