Repair of a Synthetic Abasic Site Involves Concerted Reactions of DNA Synthesis Followed by Excision and Ligation

Abstract

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.