APE1 active site residue Asn174 stabilizes the AP-site and is essential for catalysis
Preprint
Abstract
Apurinic/Apyrimidinic (AP)-sites are common and highly mutagenic DNA lesions that can arise spontaneously or as intermediates during Base Excision Repair (BER). The enzyme apurinic/apyrimidinic endonuclease 1 (APE1) initiates repair of AP-sites by cleaving the DNA backbone at the AP-site via its endonuclease activity. Here, we investigated the functional role of the APE1 active site residue N174 that contacts the AP-site during catalysis. We analyzed the effects of three rationally designed APE1 mutations that alter the hydrogen bonding potential, size, and charge of N174: N174A, N174D, and N174Q. We found impaired catalysis of the APE1 N174A and APE1 N174D mutants due to disruption of hydrogen bonding and electrostatic interactions between residue 174 and the AP-site. In comparison, the APE1 N174Q mutant was less impaired due to retaining similar hydrogen bonding and electrostatic characteristics as N174 in wild-type APE1. Structures and computational simulations further revealed that the AP-site was destabilized within the active sites of the APE1 N174A and APE1 N174D mutants due to loss of hydrogen bonding between residue 174 and the AP-site. Cumulatively, we show that N174 stabilizes the AP-site within the APE1 active site through hydrogen bonding and electrostatic interactions to enable effective catalysis. These findings highlight the importance of N174 in APE1’s function and provide new insights into the molecular mechanism by which APE1 processes AP-sites during DNA repair.Keywords
All Related Versions
- Published version: Journal of Biological Chemistry, 301 (10), 110655.
This publication has 59 references indexed in Scilit:
- Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genesDNA Repair, 2021
- APE1: A skilled nucleic acid surgeonDNA Repair, 2018
- Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complexProteins: Structure, Function, and Bioinformatics, 2018
- Oxidative DNA damage in disease—Insights gained from base excision repair glycosylase‐deficient mouse modelsEnvironmental and Molecular Mutagenesis, 2014
- Event detection and sub‐state discovery from biomolecular simulations using higher‐order statistics: Application to enzyme adenylate kinaseProteins: Structure, Function, and Bioinformatics, 2012
- DNA glycosylases: in DNA repair and beyondChromosoma, 2011
- Database of mouse strains carrying targeted mutations in genes affecting biological responses to DNA damage Version 7DNA Repair, 2006
- Oxidative DNA damage in cancer patients: a cause or a consequence of the disease development?Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 2003
- The penultimate rotamer libraryProteins: Structure, Function, and Bioinformatics, 2000
- The enzymology of apurinic/apyrimidinic endonucleasesMutation Research/DNA Repair, 1990