We developed and measured a distribution of genes broken by KillerRed fusion protein derived reactive oxygen. The highly organized DNA architecture inside of nuclei of cells is widely accepted. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. The smaller chromosomes are localized in a central nuclear position, the larger chromosomes peripherally. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and histone H2A combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light (above 400nm) avoiding damage by UV light.
The ROS activity in the cells allowed to gain information about the location of the damaged genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and certain diseases was found. Using independent KRED-lamin B1 probes we detected the following damaged genes REXO1L2P, MLL2, LAMA5, and MUC12 with the nearly identical read count numbers. (RNA exonuclease 1 homologue-like 2 pseudogene-REXO1L2P (OMIM ID 609614), Histone-lysine N-methyltransferase MLL2-MLL2 (OMIM ID 602113), Laminin subunit -5 precursor LAMA5 (OMIM ID 601033), Mucin-12 precursor MUC12 (OMIM ID 604609).
The results were published in: