Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III

Abstract

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R *HaeIII from Haemophilus aegyptius; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the HaeIII-produced AAV fragments relative to the three EcoRi fragments was determined. Using revised figures for the molecular weights of the HaeIII cleavage products of +X174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After HaeIII digestion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two moreslowly-migrating cleavage products also bore 5' 32p end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with HaeIII, and evidence that one may have a nonlinear ("rabbitear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.

Genome localization of adeno-associated virus RNA

“…This phenomenon was previously observed with the terminal Hind fragments of AAV2 DNA and was ascribed to differences in size resulting from a limited permutation in the DNA nucleotide sequence (7,21). We report elsewhere (20) that HaeIII cleavage produces two main terminal fragments (denoted as a and y, respectively) and variable amounts of a minor component (denoted as f8) from either end of the AAV DNA duplex and also from the duplex panhandle region of purified single strands. Further characterization of the BamBl and -B2 fragments will be reported in a subsequent communication.…”

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA

“…Because both plus and minus strands are encapsidated, purified AAV DNA can anneal to form duplex molecules. A variety of experiments, primarily involving restriction enzyme analysis, have demonstrated that half of the termini of duplex molecules formed in vitro have a normal double helical structure; the other half have an aberrant secondary structure (4,6,9,24). Fine mapping of the HpaII sites within the terminal repetition of AAV-2 DNA by using two methods, complete digestion and the partial digestion technique first described by Smith and Bimstiel (23), yielded paradoxical results (24).…”

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome

“…When complementary strands of AAV DNA are annealed, two types of secondary structures can result at the termini: a normal double helix, when both strands have a terminal sequence with the same orientation, or an aberrant structure, when the two strands contain terminal sequences of opposite orientation. Digestion with a restriction endonuclease whose terminal recognition sequence is within the region affected will lead to several species of terminal fragments, two from ends with a normal double helical conformation (one from each of the two possible sequences) and several more from the ends with an aberrant secondary structure (the exact number depends on the number of recognition sites within the region and the number of different conformations possible) (6,16). Appropriate restriction enzymes can thus be used to distinguish between normal and aberrant terminal structures.…”