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BCH6415-1 - CH 6415 DNA Virus Classification and...

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CH 6415 – DNA Virus Classification and Replication
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Basic Virus Structure
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Viral Strategies and Replication Cycle
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Chromosome 19 integration requires AAV Rep protein expression.
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AAV Replication FIG. 1. Model of AAV DNA replication. The boxed region illustrates the steps involved in the terminal resolution of AAV viral ends. In vitro, Rep68 is necessary and sufficient for both the site-specific endonuclease and helicase activities required for terminal resolution. The viral 3' end is indicated with an arrow. Circles depict Rep covalently attached to the viral 5' end at the terminal resolution site ( trs ).
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ITR Sequence and Genome Map
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Promoter Structure
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Decoding the AAV Genome Virology (1989) 173:120-128. Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication. Chejanovsky N, Carter BJ.
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4, 20 no AUG no Rep 52, 40
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Rep Protein Deletion Analysis J. Virol. 74:2936-2942 (2000). Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates. Michael D. Davis, Jianwen Wu, and Roland A. Owens * FIG. 1. AAV ITR hairpin DNA ("flop" configuration). The positions of the primary Rep68/78 recognition sequence (RRS) (52) and the secondary binding site for Rep68/78 (RRS') (30, 55) are within the labeled rectangles. The individual imperfect GAGC repeats of the RRS are indicated by subdivisions of its rectangle. The positions of the terminal resolution site (trs) (13) and the 3' ends of unfilled hairpin DNA and partially filled hairpin DNA are also indicated. Fifty-four base pairs at the right end of the hairpin sequence are indicated by dots. FIG. 3. Rep68/78 amino acids 3 to 200 are sufficient for trs endonuclease activity on unfilled hairpin DNA. trs endonuclease assays were performed (as detailed in the text) with the truncated fusion proteins indicated above each lane. Samples contained either no protein, 1.0 μg of MBP-LacZ (LacZ), or 1.0 μg of the fusion protein with the indicated truncation. The wild-type (W-type) lanes contained 1.0, 0.5, 0.25, or 0.125 μg of MBP- Rep68 . All reaction mixtures were incubated at 37°C for 60 min, boiled, and resolved by nondenaturing PAGE (6% polyacrylamide). The positions of the substrate and released cleavage product are indicated on the right. Each sample contained 25,000 cpm of 5' 32 P-end-labeled unfilled (A) or filled (B) AAV terminal repeat hairpin DNA. Unfilled Filled
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FIG. 5. The Y156F protein lacks trs endonuclease activity but retains DNA helicase activity. Assays were performed (as detailed in the text) with MBP-Rep68 (W-type) or the mutant fusion proteins indicated above each lane. MBP-LacZ (LacZ) was included as a negative control. Samples contained either no protein or the amount of each protein (in micrograms) indicated below each lane. (A) trs endonuclease assays. Each sample contained 25,000 cpm of 5' 32 P-end-labeled unfilled AAV hairpin DNA. All reaction mixtures were incubated at 37°C for 60 min, boiled, and resolved on a nondenaturing 6% polyacrylamide gel. The positions of the substrate and
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