Master of Science
Pharmacology & Toxicology
Lawrence F Povirk
Radiotherapy is the clinical application of the ionizing radiation to treat cancer. Ionizing radiation causes multiple modes of damage to the DNA damage such as SSBs, DSBs and modified bases such as thymine glycol. These lesions can exist as clusters in one or two helical turns of DNA. DNA double-strand breaks (DSBs) are extremely toxic to cells because they can lead to genomic rearrangements and even cell death. If base lesions accompany these DSBs, there will be a substantial hindrance for repair.
NHEJ is the primary DSB repair pathway in mammalian cells. HRR repairs single strand breaks (SSBs) or Double strand breaks (DSBs), during late S phase and G2 phase of the cell cycle, by using an undamaged copy of the DNA sequence, and is therefore largely error-free. The NHEJ pathway repairs DSBs without the requirement for sequence homology and can be error-free or error-prone, and is most active during G1 phase.
Thymine glycol (Tg) is the most common oxidation product of thymine. It is produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as ionizing radiation (IR); it is one of the predominant types of base modifications produced by ionizing radiation. Due to clustering of radiation-induced damages, many DSBs are accompanied by damaged bases such as Tg at or near the DSB ends that may interfere with subsequent gap filling and ligation. The base excision repair pathway plays a major role in removal of thymine glycol from the damaged DNA strand. During NHEJ, after synapsis by Ku and DNAPKcs and processing of the DNA ends, XRCC4/Ligase IV complex ligates the DNA. This ligase activity is promoted by the interaction of XLF/XRCC4 filament with Ligase IV.
Linearized plasmids with Tg at the 5th -Tg5 positions from the broken end were subjected to a repair assay using XRCC4-like factor (XLF)-deficient cell extracts, with or without the addition of XLF and Endonuclease III and/or ddTTP and Klenow fragments. End joining of Tg5 was compared to plasmid with Tg at third position-Tg3 in extracts. In addition, the ability of purified NHEJ proteins Ku, DNAPKcs and XRCC4/Ligase IV, to repair the Tg1 (Tg at the end), Tg2 (Tg at the second position), Tg3 and Tg5 in the presence and absence of XLF was assessed.
The data indicated that the cell extract could ligate the Tg5 plasmids only in the presence of XLF. End joining of the Tg5 was less in comparison to Tg3 with base excision repair being more active in Tg5 competing with the joining. Plasmids with Tg were treated with Endonuclease III and ddTTP to test whether the end joining occurred before or after Tg removal.
Endonuclease III and ddTTP treatment showed reduced intensity of the joined fragment suggesting that end joining occurred without removal of Tg in some cases. The extracts were not able to fill in the processed end by the BER though it has a 3′-OH which was filled in by the treatment with Klenow enzymes derived from E.coli DNA Polymerase I, following removal of extract proteins by proteolysis. When purified proteins were used to treat the plasmids, it is observed that there was increased efficiency of repair with increased distance of the Tg from the end may be due to less distortion of the ends as Tg is away from the end. While Tg1 and Tg2 required XLF presence for the repair, Tg3 and Tg5 could show a small amount of repair in its absence. XLF enhanced the repair of Tg3 and Tg5. While the repair by extract showed no repair in the same substrates without XLF, there was repair by purified proteins without XLF, suggesting there is some competition with the XRCC4/Ligase for the DSB by other proteins and XLF is required to overcome this.
In conclusion, cell extract was able to ligate the plasmid with Tg located at fifth position from the DSB but with lower efficiency compared to Tg3 plasmids. The base excision repair pathway is more functional if the modified base is far from the DSB. End joining by the purified proteins was proportional to the thymine glycol position from the DSB end. XLF was mandatory for repair of Tg1 and Tg2 by the purified proteins, and not for Tg3 and Tg5.
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