Abstract
Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, beta-pol, 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 moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase 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 endonuclease, 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 removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of beta-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.
Highlights
From the ‡Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the §Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, and the ¶Institute of Biotechnology, Center for Molecular Medicine, University of Texas Health Science Center, San Antonio, Texas 78245
The reconstituted Base excision repair (BER) system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system
These results indicate that the predominant pathway for AP site BER is AP endonuclease cleavage followed by DNA synthesis; a slow removal of the deoxyribonucleotide phosphate (dRP) moiety occurs that limits overall BER
Summary
In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase  (-pol) and DNA ligase I These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. Mammalian cells can repair abasic sites, an intermediate of BER, using at least two distinct pathways: one involving single nucleotide gap filling by DNA polymerase  (“simple” BER) and an “alternate” pathway that involves proliferating cell nuclear antigen (PCNA). To define the influence of this and other putative proteinprotein interactions on catalytic activities of enzymes that participate in simple BER, we reconstituted BER of a DNA substrate containing an AP site with three purified human enzymes: AP endonuclease, DNA polymerase , and DNA ligase I. Because the overall repair of the AP site occurred at a rate similar to that of dRP removal (dRP lyase step), we suggest that -pol dRP lyase activity could determine the choice between the simple and alternate BER pathways
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