Nathaniel Otoo

Demple Laboratory
Harvard School of Public Health


Origin of abasic sites in APE1-deficient mammalian cells

Abasic sites are one of the most frequently generated lesions in DNA. They arise either spontaneously from the hydrolysis of the N-glycosidic bonds or by the catalytic activity of DNA glycosylases that remove damaged, modified or even normal bases from DNA. Efficient removal of abasic sites from the DNA is critical to maintaining genomic integrity as these sites could be cytotoxic or mutagenic. It is believed that repair of abasic sites are initiated by APE1, an endonuclease which cleaves the phosphodiester backbone 5' to the abasic site leaving a 3'-hydroxyl group and a 5'-deoxyribose phosphate (dRP) group flanking the nucleotide gap. APE1 is believed to have other biological roles besides base excision repair (BER). Perhaps it is this multifunctional property that makes APE1 an essential protein in cells in that generation of APE1 knockout mice was attempted, only to reveal that these mice die during embryogenesis.

In order to fully understand the role of APE1 in vivo, short interference RNA (siRNA) technique was used to disrupt the expression of the protein in mammalian cells because stable APE1-deficient cell lines have not been reported. Deficiency of APE1 led to a robust accumulation of abasic sites. This may explain the observed increased apoptosis and G1 arrests in these cells since abasic sites are known to be pro-mutagenic lesions as well as strong replication blocks, important cytotoxic processes in the cell. The goal of my research is to identify the origin of these abasic sites in APE1-deficient mammalian cells. That is to ask the question: are these abasic sites originating from spontaneous depurination or from specific DNA.

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