Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Sahiti Kuppa; Elliot Corless; Colleen Caldwell; Maria Spies; Edwin Antony

doi:10.1016/j.ymeth.2024.02.006

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Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Journal article

Peer reviewed

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Sahiti Kuppa, Elliot Corless, Colleen Caldwell, Maria Spies and Edwin Antony

Methods (San Diego, Calif.), Vol.224, pp.47-53

04/2024

DOI: 10.1016/j.ymeth.2024.02.006

PMCID: PMC10960328

PMID: 38387709

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https://research.vu.nl/en/publications/f1dda63b-9c2a-4d33-96e9-d2a4bf79fcb9View

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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 (XPA ) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPA and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Nucleotide Excision Repair (NER)

Genetic code expansion (GCE)

Protein Dynamics

Replication Protein A (RPA)

Site-Specific Labeling

Xeroderma Pigmentosum group A (XPA)

DNA Binding Proteins

Fluorescence Enhancement through non canonical Amino Acids (FEncAA)

Details

Title: Subtitle: Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair
Creators: Sahiti Kuppa - Saint Louis University
Elliot Corless - Saint Louis University
Colleen Caldwell - University of Iowa
Maria Spies - University of Iowa
Edwin Antony - Saint Louis University
Resource Type: Journal article
Publication Details: Methods (San Diego, Calif.), Vol.224, pp.47-53
DOI: 10.1016/j.ymeth.2024.02.006
PMID: 38387709
PMCID: PMC10960328
NLM abbreviation: Methods
eISSN: 1095-9130
Grant note: DOI: 10.13039/100000054, name: National Cancer Institute, award: F99 CA274696, S10 OD030343; DOI: 10.13039/100000057, name: National Institute of General Medical Sciences, award: R35GM131704, R35GM149320; DOI: 10.13039/501100009404, name: MIRA Institute, University of Twente; DOI: 10.13039/100000002, name: National Institutes of Health
Language: English
Electronic publication date: 02/20/2024
Date published: 04/2024
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984560490502771

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