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Nucleic Acids Research, 2002, Vol. 30, No. 10 2124-2130
© 2002 Oxford University Press

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins

B. Pascucci, G. Maga1, U. Hübscher2, M. Bjoras3, E. Seeberg3, I. D. Hickson4, G. Villani5, C. Giordano6, L. Cellai7 and E. Dogliotti*

Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy, 1Istituto di Genetica Biochimica ed Evoluzionistica, IGBE-CNR, National Research Council, Via Abbiategrasso 207, I-27100 Pavia, Italy, 2Institut für Veterinärbiochemie, Universität Zürich, Winterthurerstrasse 190, A-8057 Zürich, Switzerland, 3Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, The National Hospital, N-0027 Oslo, Norway, 4ICRF, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK, 5Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, F-31077 Toulouse Cedex, France, 6Istituto di Chimica Biomolecolare, CNR, Sezione di Roma, Università ‘La Sapienza’, P. le A. Moro 5, I-00185 Rome, Italy and 7Istituto di Cristallografia, CNR, Sezione di Roma, PO Box 10, Monterotondo Stazione, I-00016 Rome, Italy

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) ß and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol ß were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol ß strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol ß and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

* To whom correspondence should be addressed. Tel: +39 06 4990 2580; Fax: +39 06 4990 2355; Email: dogliott{at}iss.it Present address:B. Pascucci, Istituto di Cristallografia, CNR, Sezione di Roma, PO Box 10, Monterotondo Stazione, I-00016 Rome, Italy


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