Biology subjects

Chaires, J. B.

Publications and source records attributed to Chaires, J. B..

5 recordsLinked to original sources

bioRxiv2024

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 [A]. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

biophysics↗

bioRxiv2020

The solution structures of higher-order human telomere G-quadruplex multimers

Human telomeres contain the repeat DNA sequence 5(TTAGGG), with duplex regions that are several kilobases long terminating in a 3 single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate modes proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5(TTAGGG)n, with n=8, 12, and 16, fold into multimeric structures containing the maximal number (2, 3, and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of about 34 [A]. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

biophysics↗

bioRxiv2019

Human POT1 Unfolds G-Quadruplexes by Conformational Selection

The reaction mechanism by which shelterin protein POT1 (Protection of Telomeres) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. We show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is fast, with an apparent relaxation time of 80.0 {+/-} 0.4 ms, and strong, with a binding free energy of -10.1 {+/-} 0.3 kcal mol-1. That favourable free energy arises from a large favourable enthalpy contribution of -38.2 {+/-} 0.3 kcal mol-1. In sharp contrast, the binding of POT1 to an initially folded 24 nt G-quadruplex structure is slow, with an average relaxation time of 2000-3000 s. Fluorescence, circular dichroism and analytical ultracentrifugation studies show that POT1 binding is coupled to quadruplex unfolding with a final stoichiometry of 2 POT1 molecules bound per 24 nt DNA. The binding isotherm for the POT1-quadruplex binding interaction is sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provides an excellent quantitative fit to the experimental binding data. The overall favourable free energy of the POT1-quadruplex interaction is -7.1 kcal mol-1, which arises from a balance between unfavourable free energy of +3.4 kcal mol-1 for quadruplex unfolding and a large, favorable free energy of -10.5 kcal mol-1 for POT1 binding. We show that POT1 can unfold and bind to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid or parallel), but will not interact with duplex DNA or with a parallel G-quadruplex structure formed by a c-myc promoter sequence. Finally, molecular dynamics simulations provide a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

biophysics↗

bioRxiv2018

Putting a New Spin of G4 Structure and Binding by Analytical Ultracentrifugation

Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.

biophysics↗

bioRxiv2017

Identification of putative G-quadruplex forming sequences in three manatee papillomaviruses

The Florida manatee (Trichechus manatus latirotris) is considered 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 (PVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). In this study, we identified DNA sequences with the potential to form G-quadruplex structures in all three PVs. G-quadruplex structures (G4) are guanine-rich nucleic acid sequences capable of forming secondary structures in DNA and RNA. In humans, G4 are known to regulate molecular processes such as transcription and translation. Although G4 have been identified in several viral genomes, including human PVs, no attempt has been made to identify G4 in animal PVs. We found that sequences capable of forming G4 were present on both DNA strands and across coding and non-coding regions on all PVs. The vast majority of the identified sequences would allow the formation of non-canonical structures with only two G-tetrads. The formation of one such structure was supported through biophysical analysis. Computational analysis demonstrated enrichment of G4 sequences on the reverse strand in the E2/E4 region on all manatee PVs and on the forward strand in the E2/E4 region on one genital PV. 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 PVs, G4 sequences were located near putative E2 binding sites in the non-coding region. Together, these findings suggest that G4 are likely regulatory elements in manatee PVs.\n\nAuthor summaryG-quadruplex structures (G4) are found in the DNA and RNA of many species and are known to regulate the expression of genes and the synthesis of proteins, among other important molecular processes. Recently, these structures have been identified in several viruses, including the human papillomavirus (PV). As regulatory structures, G4 are of great interest to researchers as drug targets for viral control. In this paper, we identify the first G4 sequences in three PVs infecting a non-human animal, the Florida manatee. Through computational and biophysical analysis, we find that a greater variety of sequence patterns may underlie the formation of these structures than previously identified. The sequences are found in all protein coding regions of the virus and near sites for viral replication in non-coding regions. Furthermore, the distribution of these sequences across the PV genomes supports the notion that sequences are conserved across PV types, suggesting they are under selective pressure. This paper extends previous research on G4 in human PVs with additional evidence for their role as regulators. The G4 sequences we identified also provide potential regulatory targets for researchers interested in controlling this virus in the Florida manatee, a threatened aquatic mammal.

molecular biology↗