Albany 2013: Book of Abstracts

category image Albany 2013
Conversation 18
June 11-15 2013
©Adenine Press (2012)

Kinetics of Apurinic/Apyrimidinic Endonuclease 1 (APE1) on an Authentic AP Site or an AP Site Analog

Our genomic DNA is endlessly exposed to a wide variety of exogenous and endogenous DNA damaging agents. One of the most abundant DNA lesions is an apurinic/apyrimidinic (AP) site, which in vivo, can form spontaneously or through various cellular pathways, including the repair activity of DNA glycosylase enzymes (Wilson & Barsky, 2001). Persistence of these AP sites is both highly mutagenic and cytotoxic to the cell (Loeb & Preston, 1986). AP endonuclease 1 (APE1), an Mg2+ dependent enzyme, is the major human endonuclease responsible for incising the DNA backbone at AP sites. Repair to canonical duplex DNA is then completed by DNA polymerase and DNA ligase. Recently, APE1, in conjunction with delivery of DNA damaging agents, has become a target for chemotherapeutic research with the aim to inhibit APE1 activity (Fishel & Kelley, 2007). Therefore, an understanding of APE1 activity and its molecular mechanism is essential. In vitro, the authentic AP site is highly unstable and can undergo β-elimination, leading to a strand break (Strauss, Beard, Patterson & Wilson, 1997). Due to the fragility of the AP site, stable AP site analogs, such as the reduced AP site or tetrahydrofuran (THF) site, are typically used to study APE1 (Strauss, Beard, Patterson & Wilson, 1997; Maher & Bloom, 2007). In this work we have performed the first comprehensive kinetic study of APE1 acting on the authentic AP site as well the reduced AP site and THF AP site analog. Transient-state kinetic experiments reveal that the strand incision chemistry step is fast, upwards of ~ 700 s-1,¬¬ for all substrates, making APE1 one of the fastest DNA repair enzymes. Steady-state kinetic experiments reveal for each substrate, a slow, post chemistry step limits the steady-state rate. The steady-state rate for APE1 acting on authentic AP and AP-Red substrates is highly dependent on Mg2+ concentration, while the steady-state rate for THF site was not dependent on Mg2+ concentration. This comprehensive kinetic analysis reveal differences and similarities in the way APE1 processes the authentic AP site compared to AP site analogs. Furthermore, these differences require consideration when choosing AP site analogs to study APE1.

This research has been supported by NIEHS :ES019296


    Wilson III, D.M., and Barsky, D., (2001) The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA. DNA Repair 485, 283 -307

    Loeb, L.A., and Preston, B.D., (1986) Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet. 20, 201-230

    Fishel, M.L., and Kelley, M.R., (2007) The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target. Mol. Aspects Med. 28, 375-395

    Strauss, P.R., Beard, W.A., Patterson, T.A., and Wilson, S. H. (1997) Substrate Binding by Human Apurinic/Apyrimidinic Endonuclease Indicates a Briggs-Haldane Mechanism. J. Biol. Chem. 272, 1302-1307

    Maher, R.L, and Bloom, L.B., (2007) Pre-Steady-state Kinetic Characterization of the AP Endonuclease Activity of Human AP Endonuclease 1. J. Biol. Chem. 282, 30577-30585

Kelly M. Schermerhorn
Sarah Delaney

Department of Chemistry
Brown University
Providence, RI 02912

Ph: (401) 863- 3590
Fx: (401) 863-1993