Investigation of nucleotide-mediated CPD thymine dimer photo-repair in duplex DNA: Development of a novel method to investigate the effect of flanking bases on the formation and repair of CPD thymine dimers in duplex DNA

In a primordial "RNA world," survival and self-replicating nucleic acid species within the extremely harsh environment of early Earth, especially UV radiation, imposed this prebiotic nucleic acid to evolve and acquire certain desirable features to survive and reproduce. Eventually, DNA develops as a more durable nucleic acid analog to store genetic information. The cis-syn Cyclobutane Pyrimidine Dimers (CPDs) are the most common lesion formed in cellular DNA from exposure to solar light. Although many studies have been carried out to identify the influence of DNA sequence and structure on its photochemical and photophysical properties, the molecular bases of these observations are not yet been well understood. This research has been conducted to comprehensively study the comparative impact of neighboring bases on either side of a pyrimidine pair on CPD formation and photo-repair of a pre-existing CPD in double-stranded DNA (dsDNA). The principle of our approach relies on precise blockage of Taq DNA polymerase at pyrimidine dimers (CPDs), which cause the disappearance of the sequences that contain a thymine dimer from the irradiated DNA library pool. High-throughput sequencing is then done to explore the formation and repair of CPDs over the time-course experiment at different wavelengths. We are using a random double-stranded DNA library consisting of a stretch of 10 randomized base pairs with a central thymine pair flanked by constant bases as primer binding sites for PCR amplification. The constant regions were designed to avoid two adjacent pyrimidines. The sources of UV irradiation being used in this research are a monochromatic 278 nm LED and a 365 nm LED with a triplet sensitizer. Our study is unique and unprecedented in terms of being done on quite long authentic dsDNA and many random nucleotides flanking the thymine pair or dimer, being studied at once. Besides, we use both UVC and UVA monochromatic LEDs to create/repair CPD dimers, which is more comparable to CPD formation due to solar exposure in biological systems. All possible sequences up to 5 nucleotides on both 5' and 3' sides are being studied and we compared our data with the known UV hot spots in the human genome.