Abu, M.; (2005) Kinetic study of human thymine DNA glycosylase and examination of its interaction with apurinic endonuclease 1 in base excision repair. Doctoral thesis , University of London.

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Abstract

In the human genome, cytosine is exclusively methylated at CpG sequences to give 5-methylcytosine. The C4-amino group of 5-methylcytosine is susceptible to spontaneous hydrolysis, generating thymine in a G T mismatch. Thymine DNA glycosylase (TDG) detects the mismatch and cleaves the glycosidic bond of thymine. Then apyrimidinic/apurinic endonuclease 1 (APEX1) displaces TDG from the abasic site and cuts the phosphodiester bond 5' to the abasic site in base excision repair. The kinetic parameters, K<i and k2, for TDG acting on thymine and ethenocytosine substrates, were measured. The excision of both substrates was very dependent upon the base 5' to the mismatched guanine. TDG has a strong preference for both thymine and ethenocytosine in a CpG sequence. This is understandable for thymine since G T mismatches arise in this context. However, ethenocytosine is not exclusively formed at CpG sequences. The catalytic step (k2) for CpG-T was six-fold faster than CpG-eC, but was bound, as shown by Kd, approximately 800-fold less tightly by TDG. This large difference in Kd is probably due to the unstable structure of G sC, which allows TDG to easily flip ethenocytosine out of the DNA. Thus, the reaction of TDG with ethenocytosine in vitro is misleading. This, together with the sequence dependence of ethenocytosine excision by TDG, means that TDG cannot be the main ethenocytosine-DNA glycosylase in vivo. The mechanism of TDG displacement by APEX1 was examined by looking at the involvement of the DNA and by looking for protein-protein interactions between TDG and APEX1. Although the DNA is not absolutely required for the displacement of TDG by APEX1, a weak/transient protein-protein interaction was detected between TDG and APEX1 using a pull-down assay. These results suggest that APEX1 transiently interacts with TDG bound to abasic DNA to induce a conformational change in TDG that dissociates it from the abasic site.

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