Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity.

ABSTRACT:

Background

Endonuclease G (EndoG), a member of DNA/RNA nonspecific betabetaalpha-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.

Methods

To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies.

Results

Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A

SUBMITTER: Wu SL 

PROVIDER: S-EPMC2653514 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature