期刊
BIOCHEMISTRY
卷 52, 期 43, 页码 7669-7677出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi401218r
关键词
-
资金
- National Institute of Environmental Health Sciences [R01ES019296]
Apurinic/apyrimidinic endonuclease 1 (APE1) is an Mg2+-dependent enzyme responsible for incising the DNA backbone 5' to an apurinic/apyrimidinic (AP) site. Here, we use rapid quench flow (RQF) techniques to provide a comprehensive kinetic analysis of the strand-incision activity (k(chemistry)) of APE1 acting on an authentic AP site along with two widely used analogs, a reduced AP site and a tetrahydrofuran (THF) site. In the presence of biologically relevant Mg2+, APE1 incises all three substrates at a rate faster than the resolution of the RQF, >= 700 s(-1). To obtain quantitative values of k(chemistry) and to facilitate a comparison of the authentic substrate versus the substrate analogs, we replaced Mg2+ with Mn2+ or Ni2+ or introduced a mismatch 5' to the lesion site. Both strategies were sufficient to slow k(chemistry) and resulted in rates within the resolution of the RQF. In all cases where quantitative rates were obtained, k(chemistry) for the reduced AP site is indistinguishable from the authentic AP site. Notably, there is a small decrease, similar to 1.5-fold, in k(chemistry) for the THF site relative to the authentic AP site. These results highlight a role in strand incision for the C1' oxygen of the AP site and warrant consideration when designing experiments using substrate analogs.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据