| Chapter title |
Detection of oriC-Independent Replication in Escherichia coli Cells
|
|---|---|
| Chapter number | 9 |
| Book title |
DNA Topoisomerases
|
| Published in |
Methods in molecular biology, January 2018
|
| DOI | 10.1007/978-1-4939-7459-7_9 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7458-0, 978-1-4939-7459-7
|
| Authors |
Makisha Martel, Aurélien Balleydier, Julien Brochu, Marc Drolet |
| Abstract |
In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2'-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor® 488 dye is conjugated to EdU by the click-iT® reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy. |
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Geographical breakdown
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|---|---|---|
| Unknown | 7 | 100% |
Demographic breakdown
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|---|---|---|
| Student > Master | 1 | 14% |
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| Researcher | 1 | 14% |
| Unknown | 4 | 57% |
| Readers by discipline | Count | As % |
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| Unknown | 4 | 57% |