It has been hypothesized that G-quadruplexes can sequester the 3′ end of
the telomere and prevent it from being extended by telomerase. Here we purify and
characterize stable, conformationally homogenous human telomeric G-quadruplexes, and
demonstrate that human telomerase is able to extend parallel, intermolecular
conformations in vitro. These G-quadruplexes align correctly with the RNA
template of telomerase, demonstrating that at least partial G-quadruplex resolution
is required. A highly purified preparation of human telomerase retains this
extension ability, establishing that the core telomerase enzyme complex is
sufficient for partial G-quadruplex resolution and extension. The parallel-specific
G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in
telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of
telomeric G-quadruplexes in vivo. The ability of telomerase to partially
unwind, extend and localize to these structures implies that parallel telomeric
G-quadruplexes may play an important biological role.
Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
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