Rep-mediated nicking of the adeno-associated virus origin requires two biochemical activities, DNA helicase activity and transesterification.
The single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trs sequence necessary for Rep cleavage, 3'-CCGGT/TG-5', and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at the trs. Interestingly, Rep nicking on a TR substrate that fixes this trs stem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trs and extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function.
Brister
J R
JR
Department of Molecular Genetics, University of Florida Gene Therapy Center, College of Medicine, Gainesville, Florida 32610, USA.
Muzyczka
N
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D004951:Esterification; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018741:Replication Origin; D020079:Terminal Repeat Sequences / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D000255:Adenosine Triphosphate; D004720:Endonucleases; D004265:DNA Helicases