| Chapter title |
Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase
|
|---|---|
| Chapter number | 14 |
| Book title |
Mitochondrial DNA
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3040-1_14 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3039-5, 978-1-4939-3040-1
|
| Authors |
Fernando A. Rosado-Ruiz, Minyoung So, Laurie S. Kaguni |
| Abstract |
The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 3 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 1 | 33% |
| Student > Doctoral Student | 1 | 33% |
| Student > Master | 1 | 33% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 2 | 67% |
| Agricultural and Biological Sciences | 1 | 33% |