Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Substrate Binding, Catalytic Activity and Product Release by Human 8-Oxo-7,8-dihydroguanine Glycosylase (hOGG1) are Modulated by the Structural Context of 8-Oxo-7,8-dihydroguanine in a CAG Trinucleotide Repeat Sequence
The DNA repair protein human 8-oxo-7,8-dihydroguanine glycosylase (hOGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized guanine base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA (1). Interestingly, OGG1 has been implicated in expansion of the trinucleotide repeat (TNR) sequence CAG/CTG and this expansion represents the molecular basis of several neurodegenerative disorders (2). Furthermore, in addition to the duplex conformation, CAG/CTG sequences have been shown to adopt non-B conformations such as stem-loop hairpins (3). Via a long-patch BER (LP-BER) pathway it has been shown in vitro that the ability of these repeat regions to form hairpins during BER can result in a flap that is refractory to flap endonuclease 1 (FEN1). Expansion of the TNR DNA is a consequence of the persistence of a trapped hairpin that can be ligated into the duplex (4,5).
We reported previously that hairpins that may form during this BER expansion mechanism contain hot spots for oxidative damage when treated with peroxynitrite (ONOO-) (6). Therefore, TNR substrates containing site-specifically incorporated 8-oxoG were then synthesized to define the kinetic parameters of hOGG1 activity on duplex and hairpin structures. In this work we first used an electrophoretic mobility shift assay to determine the KD for hOGG1 binding to hairpin and duplex substrates in which the position of the 8-oxoG was varied. Second, the rate at which hOGG1 catalyzes excision of 8-oxoG was quantified by performing single-turnover experiments. Third, multiple-turnover experiments were used to define the rate of product release for hOGG1 acting on the hairpin and duplex substrates. As a benchmark for hOGG1 activity, the data obtained for the TNR substrates were compared to those obtained for a mixed-sequence duplex.
We find that hOGG1 binding, activity and product release for TNR duplexes is indistinguishable from the mixed-sequence control, indicating the BER can be initiated by hOGG1 just as efficiently in TNR regions of DNA as elsewhere in the genome. Interestingly, the activity of hOGG1 is modulated by the structure of the DNA substrate. For the hairpin substrates, hOGG1 has a reduced affinity, excises 8-oxoG at a slower rate, and releases the DNA product faster as compared to the corresponding TNR duplex substrates.
Daniel A. Jarem
Department of Chemistry