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DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.
- Source :
-
Nucleic acids research [Nucleic Acids Res] 2019 Sep 05; Vol. 47 (15), pp. 7767-7780. - Publication Year :
- 2019
-
Abstract
- A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.<br /> (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Subjects :
- Base Sequence
Binding Sites
Cloning, Molecular
DNA genetics
DNA metabolism
DNA Damage
DNA-(Apurinic or Apyrimidinic Site) Lyase genetics
DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism
Electron Spin Resonance Spectroscopy
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Genetic Vectors chemistry
Genetic Vectors metabolism
Humans
Molecular Dynamics Simulation
Nucleic Acid Conformation
Oligonucleotides metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Substrate Specificity
DNA chemistry
DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry
Oligonucleotides chemistry
Spin Labels chemical synthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1362-4962
- Volume :
- 47
- Issue :
- 15
- Database :
- MEDLINE
- Journal :
- Nucleic acids research
- Publication Type :
- Academic Journal
- Accession number :
- 31329919
- Full Text :
- https://doi.org/10.1093/nar/gkz620