Authors: Jennifer L. Furman, Pui-Wing Mok, Ahmed H. Badran, and Indraneel Ghosh*
Journal: Journal of the American Chemical Society
Affiliation: Department of Chemistry and Biochemistry, University of Arizona
This article documents the development of a protein-based approach to recognizing specific types of DNA damage in native DNA. Though several methods exist for the sensing of DNA damage, often times, only general DNA damage can be detected rather than specific lesion types and the DNA must be fragmented and quantized by chromatographic or mass spectrometric techniques. Therefore, these researchers sought to specifically sense DNA with oxidative or UV damage using proteins that fluoresce when bound directly to the damaged base pairs in situ.
To do this, they designed a sensor with three protein domains, a light emitting signal domain and two DNA binding domains. The signal domain utilizes a split-luciferase which is a bioluminescent enzyme that, when assembled, emits light as it oxidizes a pigment substrate, luciferin. The two pieces of the luciferase are bound by a flexible linker to two separate protein structures known to bind DNA in specific ways. A methyl-CpG binding domain (MBD) is used to attach one fragment of the luciferase firmly to the DNA by seeking out methylated cytosine residues that are common in mammalian DNA. The other luciferase fragment is attached to a damage binding protein.
This research utilizes two damage-binding proteins that can detect two separate forms of DNA damage. The first sensor uses oxoguanine glycosylase 1 (OGG1). OOG1 is a protein that is regularly associated with the natural base excision repair of 8-oxoguanine, a product of oxidative DNA damage. The OOG1 utilized in this system, however, is a point mutant that lacks the glycosylase/AP lyase activity so it will bind tightly to the damage site without excising the damaged bases. The second sensor uses Damaged-DNA binding protein 2 (DBB2). DBB2 is another protein associated with natural DNA damage repair systems and is known to bind to pyrimidine dimers and similar DNA lesions that are caused by UVC light.
In the experiments, the sensors are produced using cell-free translation system. Cell lysate is added to a solution of mRNA encoding for the protein domains. After the sensors have been assembled, the DNA sample is added. The MBD domain of the sensor binds the methylated cytosine residues thus localizing the first luciferase fragment to the DNA helix so that it can recombine with a nearby complementary fragment once the damage binding domain has located and bound to a nearby damage site. Once the sensor is in place, luciferin is added to the solution and the subsequent light emission is measured by a luminometer.
To validate their sensors, they evaluated the signal quality in four types of DNA: Oligonucleotide sequences, plasmid DNA, mammalian genomic DNA, and in HeLa cells. These DNA samples were systematically subjected to UVC light or to CuCl2/H2O2 oxidation and then exposed to the corresponding sensor. In each case, the sensor signal was significantly higher in the damaged DNA than in the control DNA. To assess the limits of specificity, the signal strength was observed for the treatment of UVC or oxidized DNA with the opposite sensor. The OGG1 sensor produced a greater signal for UVC exposed DNA when compared to the control, though the signal was much lower than that of the oxidized DNA. The DDB2 DNA also produced a signal when used with the oxidized DNA. This result suggests that further optimization is needed for the structure of the sensors in order to ensure specificity of DNA lesions. Overall, this method is successful in using conditionally activated split-protein sensors to identify sites of DNA damage. This technology will certainly be useful in the elucidation of DNA damage and repair mechanisms.