Methods in Enzymology 2006-01-01

Analysis of base excision DNA repair of the oxidative lesion 2-deoxyribonolactone and the formation of DNA-protein cross-links.

Jung-Suk Sung, Bruce Demple

Index: Meth. Enzymol. 408 , 48-64, (2006)

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Abstract

DNA base lesions arising from oxidation or alkylation are processed primarily by the base excision repair pathway (BER). The damaged bases are excised by DNA N-glycosylases, which generate apurinic/apyrimidinic (AP) sites; AP sites produced by hydrolytic decay of DNA or the spontaneous loss of damaged bases are also processed by BER. Free radicals produce various types of abasic lesions as oxidative damage. This chapter focuses on the analysis of DNA repair and other reactions that occur with the lesion 2-deoxyribonolactone (dL), which has received much attention recently. DNA substrates with site-specific dL lesions are generated by photolysis of a synthetic precursor residue; both small oligonucleotide and plasmid-based substrates can be produced. The dL residue is readily incised by AP endonucleases such as the mammalian Ape1 protein, which would bring the lesion into BER. However, the second enzyme of the canonical BER pathway, DNA polymerase beta, instead of excising Ape1-incised dL, forms a stable DNA-protein cross-link with the lesion. Such cross-links are analyzed by polyacrylamide gel electrophoresis. Incubation of Ape1-incised dL substrates with mammalian cell-free extracts shows that other proteins can also form such cross-links, although DNA polymerase beta appears to be the major species. This chapter presents methods for analyzing the extent of DNA repair synthesis (repair patch size) associated with dL in whole cell extracts. These analyses show that dL is processed nearly exclusively by the long patch BER pathway, which results in the repair synthesis of two or more nucleotides.