MAMMALIAN ABASIC SITE BASE EXCISION-REPAIR - IDENTIFICATION OF THE REACTION SEQUENCE AND RATE-DETERMINING STEPS

Base excision repair (BER) is one of the cellular defense mechanisms r epairing damage to nucleoside 5'-monophosphate residues in genomic DNA . This repair pathway is initiated by spontaneous or enzymatic N-glyco sidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) s ite in double-stranded DNA. Class II AP endonuclease, deoxyribonucleot ide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities co mplete repair of the AP site. In mammalian cell nuclear extract, BER c an be mediated by a macromolecular complex containing DNA polymerase b eta (beta-pol) and DNA ligase I. These two enzymes are capable of cont ributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitr o using the following purified human proteins:AP endonuclease, beta-po l, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' ''nicking'' of the AP site-containing DNA strand by AP endonuclease , beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiet y in the gap. Removal of the (dRP flap is strictly required for DNA li gase I to seal the resulting nick, Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonucle ase, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system, Instead, the rate-limiting step in the reconstituted system was found to be remova l of dRP tie. dRP lyase), catalyzed by the amino-terminal domain of be ta-pol, This work is the first to measure the rate of BER in an in vit ro reaction. The potential significance of the dRP-containing intermed iate in the regulation of BER is discussed.