Mammalian abasic site base excision repair - Identification of the reaction sequence and rate-determining steps

被引:346
作者
Srivastava, DK
Vande Berg, BJ
Prasad, R
Molina, JT
Beard, WA
Tomkinson, AE
Wilson, SH
机构
[1] NIEHS, Struct Biol Lab, NIH, Res Triangle Pk, NC 27709 USA
[2] Univ Texas, Med Branch, Dept Human Biol Chem & Genet, Galveston, TX 77555 USA
[3] Univ Texas, Hlth Sci Ctr, Ctr Mol Med, Inst Biotechnol, San Antonio, TX 78245 USA
关键词
D O I
10.1074/jbc.273.33.21203
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 tie. 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.
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收藏
页码:21203 / 21209
页数:7
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