Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein
Document Type
Article
Publication Date
1-1-2016
Subject Area
DNA Helicases (metabolism); DNA Polymerase gamma; DNA Replication (genetics); DNA, Mitochondrial (genetics); DNA, Single-Stranded (genetics); DNA-Binding Proteins (genetics); DNA-Directed DNA Polymerase (genetics, metabolism); Electrophoretic Mobility Shift Assay (methods); Escherichia coli (genetics, metabolism); Humans; Mitochondria (genetics, metabolism); Mitochondrial Proteins (genetics)
Abstract
The mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the function of replisome components at the mitochondrial replication fork. In recent years, it has been demonstrated that mtSSB stimulates the activities of DNA polymerase γ (Pol γ) and mitochondrial DNA (mtDNA) helicase in a concentration-dependent manner. Here we present a new approach to purify the human mtSSB and our standard assays to evaluate its biochemical properties, including a Gel Mobility Shift Assay (GMSA) to assess single-stranded DNA (ssDNA) binding activity, and an assay to assess SSB stimulation of Pol γ activity.
Publication Title
Methods in molecular biology (Clifton, N.J.)
Volume
1351
First Page
211
Last Page
22
Digital Object Identifier (DOI)
10.1007/978-1-4939-3040-1_16
PubMed ID
26530685
E-ISSN
1940-6029
Language
eng
Citation Information
Ciesielski, Grzegorz L.; Rosado-Ruiz, Fernando A.; and Kaguni, Laurie S., "Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein" (2016). UNF Faculty Research and Scholarship. 3290.
https://digitalcommons.unf.edu/unf_faculty_publications/3290