An Improved Model for the hTERT Promoter Quadruplex

<div><p>Mutations occur at four specific sites in the hTERT promoter in >75% of glioblastomas and melanomas, but the mechanism by which the mutations affect gene expression remains unexplained. We report biophysical computational studies that show that the hTERT promoter sequence forms a novel G-quadruplex structure consisting of three contiguous, stacked parallel quadruplexes. The reported hTERT mutations map to the central quadruplex within this structure, and lead to an alteration of its hydrodynamic properties and stability.</p></div

The hTERT promoter sequence and proposed structure.

<p><i>Top</i>: Sequence of the hTERT core promoter with the positions of mutations observed in melanomas shown in red. <i>Bottom</i>: Previously proposed structure of the hTERT core promoter (5). Adapted with permission from (5).</p

Oligodeoxynucleotides used in this study.

<p>Oligodeoxynucleotides used in this study.</p

Results of sedimentation velocity experiments of hTERT promoter sequences in solution.

<p>The distribution of sedimentation coefficients, corrected for viscosity and temperature, are shown, revealing a major species with S_20,w of 4.05+/−0.04 for the folded wild-type sequence. S_20,w values for folded mutant sequences are slightly but significantly reduced to 3.5 - 3.6 S_20,w.</p

Circular dichroism studies of hTERT and mutant sequences.

<p>(A) Molar circular dichroism (Δε) of the hTERT promoter sequence in solution (black). The spectrum predicted for the structure shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115580#pone-0115580-g001" target="_blank">Fig. 1</a> is shown in red. The experimentally observed spectrum for the parallel quadruplex structure formed by the c-myc promoter sequence variant 1XAV is shown in blue. (B). Molar circular dichroism of the folded wild-type and mutant sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115580#pone-0115580-g001" target="_blank">Fig. 1</a>.</p

Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.

<p>Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.</p

Identification of G-quadruplex forming sequences in three manatee papillomaviruses

<div><p>The Florida manatee (<i>Trichechus manatus latirotris</i>) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.</p></div

Identification of G-quadruplex forming sequences in three manatee papillomaviruses - Fig 2

<p><b>Coding and non-coding regions aligned across TmPV1 (outer), TmPV3 (middle), and TmPV4 (inner) to illustrate G4 sequences occurring at the same location within regions on the forward (left) and reverse (right) DNA strands.</b> Blue arrows indicate G4 sequences at the same location on TmPV3 and TmPV4. Red arrows indicate G4 sequences at the same location on all three genomes.</p

Hydrodynamic properties of the major TmPV sequences^*.

<p>Hydrodynamic properties of the major TmPV sequences^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195625#t002fn001" target="_blank">*</a>}.</p

Molecular dynamics-derived model of the four stacked contiguous G-quadruplexes for sequence TmPV4-2.

<p>Individual G-quadruplex structures appear from top to bottom in orange, magenta, yellow, and red. G-tetrads are identified in elemental colors in Chimera <b>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195625#pone.0195625.ref088" target="_blank">88</a>]</b>.</p