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Nucleic Acids Research, 2000, Vol. 28, No. 11 2207-2213
© 2000 Oxford University Press

Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding

D. G. Rothwell, B. Hang1, M. A. Gorman2, P. S. Freemont2, B. Singer1 and I. D. Hickson*

Imperial Cancer Research Fund Laboratories, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK, 1Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA and 2Protein Structure and Function Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity ~500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1–D210E protein has a 3000-fold reduced Kcat for AP site cleavage, but an unchanged Km. Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1–substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.

* To whom correspondence should be addressed. Tel: +44 1865 222417; Fax: +44 1865 222431; Email: hickson@icrf.icnet.uk Present address: D. G. Rothwell, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, UK The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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