Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly used substrate analogs: the effect of diverse metal ions and base mismatches.
Biochemistry
; 52(43): 7669-77, 2013 Oct 29.
Article
em En
| MEDLINE
| ID: mdl-24079850
ABSTRACT
Apurinic/apyrimidinic endonuclease 1 (APE1) is an Mg²âº-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 Mg²âº, APE1 incises all three substrates at a rate faster than the resolution of the RQF, ≥700 s⻹. To obtain quantitative values of k(chemistry) and to facilitate a comparison of the authentic substrate versus the substrate analogs, we replaced Mg²âº with Mn²âº or Ni²âº 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Modelos Moleculares
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Pareamento Incorreto de Bases
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DNA Liase (Sítios Apurínicos ou Apirimidínicos)
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Magnésio
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Manganês
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Níquel
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article