Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.
J Phys Chem B
; 125(30): 8351-8361, 2021 08 05.
Article
en En
| MEDLINE
| ID: mdl-34309392
ABSTRACT
The combination of DNA force spectroscopy and polarization microscopy of fluorescent DNA intercalator dyes can provide valuable insights into the structure of DNA under tension. These techniques have previously been used to characterize S-DNA-an elongated DNA conformation that forms when DNA overstretches at forces ≥ 65 pN. In this way, it was deduced that the base pairs of S-DNA are highly inclined, relative to those in relaxed (B-form) DNA. However, it is unclear whether and how topological constraints on the DNA may influence the base-pair inclinations under tension. Here, we apply polarization microscopy to investigate the impact of DNA pulling geometry, torsional constraint, and negative supercoiling on the orientations of intercalated dyes during overstretching. In contrast to earlier predictions, the pulling geometry (namely, whether the DNA molecule is stretched via opposite strands or the same strand) is found to have little influence. However, torsional constraint leads to a substantial reduction in intercalator tilting in overstretched DNA, particularly in AT-rich sequences. Surprisingly, the extent of intercalator tilting is similarly reduced when the DNA molecule is negatively supercoiled up to a critical supercoiling density (corresponding to â¼70% reduction in the linking number). We attribute these observations to the presence of P-DNA (an overwound DNA conformation). Our results suggest that intercalated DNA preferentially flanks regions of P-DNA rather than those of S-DNA and also substantiate previous suggestions that P-DNA forms predominantly in AT-rich sequences.
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1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN
Idioma:
En
Revista:
J Phys Chem B
Asunto de la revista:
QUIMICA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos