Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII

Genes
Olga A KladovaN A Kuznetsov

Abstract

Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and β, δ-elimination of 3'- and 5'-phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5'-phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does....Continue Reading

References

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Citations

Oct 18, 2018·Nucleic Acids Research·Alexandra A KuznetsovaNikita A Kuznetsov
Aug 31, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Danila A IakovlevOlga S Fedorova
Aug 28, 2021·International Journal of Molecular Sciences·Alexandra A KuznetsovaNikita A Kuznetsov
Oct 22, 2019·The Journal of Physical Chemistry. B·Irina V AlekseevaNikita A Kuznetsov

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Methods Mentioned

BETA
electrophoresis
Fluorescence
X-ray
Assay

Software Mentioned

Pro Analyzer
DynaFit
Gel

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