Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.

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Version: author
Serval ID
serval:BIB_9C7D202B44D3
Type
Article: article from journal or magazin.
Collection
Publications
Title
Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.
Journal
Nucleic Acids Research
Author(s)
Kostyrko K., Mermod N.
ISSN
1362-4962 (Electronic)
ISSN-L
0305-1048
Publication state
Published
Issued date
2016
Peer-reviewed
Oui
Volume
44
Number
6
Pages
e56
Language
english
Abstract
DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.
Pubmed
Open Access
Yes
Create date
21/01/2016 12:25
Last modification date
20/08/2019 16:03
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