Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA

Jennifer L. Furman, Pui Wing Mok, Ahmed H. Badran, Indraneel Ghosh

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.

Original languageEnglish (US)
Pages (from-to)12518-12527
Number of pages10
JournalJournal of the American Chemical Society
Volume133
Issue number32
DOIs
StatePublished - Aug 17 2011

Fingerprint

DNA Damage
DNA
Luciferases
Sensors
Firefly Luciferases
Pyrimidine Dimers
Cytosine
DNA-Binding Proteins
HeLa Cells
Oligonucleotides
Mutagenesis
DNA Repair
Reactive Oxygen Species
Carcinogenesis
Plasmids
Cell Death
Nucleotides
Genome
Light
8-hydroxyguanine

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA. / Furman, Jennifer L.; Mok, Pui Wing; Badran, Ahmed H.; Ghosh, Indraneel.

In: Journal of the American Chemical Society, Vol. 133, No. 32, 17.08.2011, p. 12518-12527.

Research output: Contribution to journalArticle

Furman, Jennifer L. ; Mok, Pui Wing ; Badran, Ahmed H. ; Ghosh, Indraneel. / Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA. In: Journal of the American Chemical Society. 2011 ; Vol. 133, No. 32. pp. 12518-12527.
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