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Detecting ultraviolet damage in single DNA molecules by atomic force microscopy.

Publication ,  Journal Article
Jiang, Y; Ke, C; Mieczkowski, PA; Marszalek, PE
Published in: Biophysical journal
September 2007

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.

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Published In

Biophysical journal

DOI

EISSN

1542-0086

ISSN

0006-3495

Publication Date

September 2007

Volume

93

Issue

5

Start / End Page

1758 / 1767

Related Subject Headings

  • Viral Proteins
  • Ultraviolet Rays
  • Sensitivity and Specificity
  • Plasmids
  • Microscopy, Atomic Force
  • Escherichia coli
  • Electrophoresis, Agar Gel
  • Dose-Response Relationship, Radiation
  • Deoxyribonuclease (Pyrimidine Dimer)
  • Deoxyribodipyrimidine Photo-Lyase
 

Citation

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ICMJE
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Jiang, Y., Ke, C., Mieczkowski, P. A., & Marszalek, P. E. (2007). Detecting ultraviolet damage in single DNA molecules by atomic force microscopy. Biophysical Journal, 93(5), 1758–1767. https://doi.org/10.1529/biophysj.107.108209
Jiang, Yong, Changhong Ke, Piotr A. Mieczkowski, and Piotr E. Marszalek. “Detecting ultraviolet damage in single DNA molecules by atomic force microscopy.Biophysical Journal 93, no. 5 (September 2007): 1758–67. https://doi.org/10.1529/biophysj.107.108209.
Jiang Y, Ke C, Mieczkowski PA, Marszalek PE. Detecting ultraviolet damage in single DNA molecules by atomic force microscopy. Biophysical journal. 2007 Sep;93(5):1758–67.
Jiang, Yong, et al. “Detecting ultraviolet damage in single DNA molecules by atomic force microscopy.Biophysical Journal, vol. 93, no. 5, Sept. 2007, pp. 1758–67. Epmc, doi:10.1529/biophysj.107.108209.
Jiang Y, Ke C, Mieczkowski PA, Marszalek PE. Detecting ultraviolet damage in single DNA molecules by atomic force microscopy. Biophysical journal. 2007 Sep;93(5):1758–1767.
Journal cover image

Published In

Biophysical journal

DOI

EISSN

1542-0086

ISSN

0006-3495

Publication Date

September 2007

Volume

93

Issue

5

Start / End Page

1758 / 1767

Related Subject Headings

  • Viral Proteins
  • Ultraviolet Rays
  • Sensitivity and Specificity
  • Plasmids
  • Microscopy, Atomic Force
  • Escherichia coli
  • Electrophoresis, Agar Gel
  • Dose-Response Relationship, Radiation
  • Deoxyribonuclease (Pyrimidine Dimer)
  • Deoxyribodipyrimidine Photo-Lyase