Journal article
Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER)
Methods in enzymology, Vol.661, pp.139-154
2021
DOI: 10.1016/bs.mie.2021.09.004
PMID: 34776210
Abstract
A detailed understanding of how homologous recombination proceeds at the molecular level in vivo requires the ability to detect in real time the appearance of specific intermediates of DNA repair. The most detailed analysis of double-strand break (DSB) repair in eukaryotes has come from the study of budding yeast, using an inducible site-specific HO endonuclease to initiate recombination synchronously in nearly all cells of the population. Polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) methods have been used to visualize the timing of the DSB, its resection by 5' to 3' exonucleases, the binding of the Rad51 recombinase and the pairing of the Rad51 filament with a homologous donor sequence. PCR has also been used to identify the next key step: the initiation of new DNA synthesis to extend the invading stand and copy the donor template. In break-induced replication (BIR), there appears to be a very long delay between strand invasion and this primer extension step. Here we describe an alternative method, an assay for monitoring BIR elongation rate (AMBER) based on digital droplet PCR that yields a much earlier time of initial DNA synthesis. We suggest that previous methods have failed to recover the initial long, single-stranded primer extension product that is readily detected by AMBER.
Details
- Title: Subtitle
- Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER)
- Creators
- Liping Liu - Department of Biology, University of Iowa, Iowa City, IA, United StatesNeal Sugawara - Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United StatesAnna Malkova - Department of Biology, University of Iowa, Iowa City, IA, United States. Electronic address: anna-malkova@uiowa.eduJames E Haber - Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States. Electronic address: haber@brandeis.edu
- Resource Type
- Journal article
- Publication Details
- Methods in enzymology, Vol.661, pp.139-154
- DOI
- 10.1016/bs.mie.2021.09.004
- PMID
- 34776210
- ISSN
- 0076-6879
- eISSN
- 1557-7988
- Grant note
- DOI: 10.13039/100000002, name: National Institutes of Health, award: R01CA232425, R35GM127006, R35GM127029
- Language
- English
- Date published
- 2021
- Academic Unit
- Biology
- Record Identifier
- 9984217525602771
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