Enzymatic bypass of G-quadruplex structures containing oxidative lesions

The function of many DNA processing enzymes involves sliding along the double helix or individual DNA strands. Stable secondary structures in the form of G-quadruplexes are difficult for such enzymes to bypass. We used a polymerase stop assay to determine which structural features of the human telomeric and the BCL2 promoter G-quadruplexes can stall progression of the Klenow fragment. Primer extension profiles revealed that G-quartets are effective roadblocks for the Klenow fragment, while auxiliary base pairs can be easily bypassed. Furthermore, we utilized 8-oxoguanine to simulate oxidative damage in G-rich regions and determine the effects on enzyme bypass. In rare cases, oxidative lesions reduce the level of G-quadruplex bypass. In general, however, oxidative lesions reduce G-quadruplex stability and facilitate bypassing of such G-rich regions, especially if the lesion persists in unfolding intermediates. Our findings using Klenow fragment can be extrapolated to other G-quadruplex forming sequences and enzymes that utilise a clamp-like structure to slide along DNA and are involved in processes such as gene expression regulation and telomere maintenance.