Book of Abstracts: Albany 2005
Replication Initiator Assembly of the Adeno-Associated Virus
Adeno-associated virus (AAV), a commonly used vector for gene therapy, has a linear ∼4.7 kb single-stranded DNA genome which contains only two ORFs, rendering it one of the smallest of all known viruses. One ORF encodes for the Rep proteins (Rep78, Rep68, Rep52, and Rep40) required for replication, and the other encodes for capsid proteins. The ∼145 bases at each end of the genome contain palindromic sequences known as inverted terminal repeats that permit each end to fold into a distinctive DNA structure characterized by a three-way DNA junction. These ends serve as the viral origins of replication.
The Rep proteins have myriad biochemical and enzymatic properties including site-specific DNA binding, site-specific DNA strand cleavage, and an ATP-dependent helicase activity. The longer Rep proteins, Rep68 and Rep68, bind to the inverted terminal repeats and carry out an intricate series of reactions required to initiate viral genome replication.
We have recently solved the structure of the Rep endonuclease domain, alone and in complex with two regions of the viral origin of replication. We have also determined the structure of the Rep helicase domain. These allow us to begin to assemble a three-dimensional picture of the nucleoprotein complexes that form at various stages during replication initiation. We propose that the initial binding of six Rep molecules at the tetranucleotide repeats that constitute the Rep Binding Site (RBS) is followed by specific binding to the tip of one of the hairpin arms of the three-way DNA junction. This second binding event initiates the assembly of the helicase domains into a hexameric ring encircling the DNA; the subsequent unwinding of DNA around the terminal resolution site is needed to generate the single-stranded form that can be recognized by the Rep endonuclease. Cleavage at the terminal resolution site liberates the free 3'-OH group that is used as the primer for template-directed synthesis during replication.
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