Abstract：
: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, ~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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Schermerhorn KM,Delaney Sdoi
10.1021/bi401218rsubject
Has Abstractpub_date
2013-10-29 00:00:00pages
7669-77issue
43eissn
0006-2960issn
1520-4995journal_volume
52pub_type
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