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Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.
Kuppa, Sahiti; Corless, Elliot; Caldwell, Colleen C; Spies, Maria; Antony, Edwin.
Afiliação
  • Kuppa S; Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
  • Corless E; Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
  • Caldwell CC; Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
  • Spies M; Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
  • Antony E; Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA. Electronic address: edwin.antony@health.slu.edu.
Methods ; 224: 47-53, 2024 Apr.
Article em En | MEDLINE | ID: mdl-38387709
ABSTRACT
Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Dano ao DNA / Reparo do DNA Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Dano ao DNA / Reparo do DNA Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article