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

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.

MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

“…We have recently constructed a MMEJ‐specific GFP reporter assay, based on principles analogous to the HR and NHEJ reporter plasmids used above (Kostyrko and Mermod, ). Interestingly, the use of this reporter in CHO cells revealed that the majority of episomal DSBs were not re‐joined by a simple MMEJ pathway.…”

“…S3). Seventy percent of the analyzed repair products had no pre‐existing microhomology indicative of MMEJ, but they displayed direct or inverted repeat sequences associated SD‐MMEJ, up‐ or downstream of the repaired junction (Kostyrko and Mermod, ). We thus concluded that plasmid‐to‐plasmid joining relies mostly on a SD‐MMEJ pathway potentially involving DNA polymerase θ and Ligase III, and that the simple MMEJ mechanism is seldom used.…”

“…These later processes are often obscured by the main repair mechanisms, which may predominate in normal cells. Furthermore, their components are still poorly characterized and there was no simple assay to specifically detect them, rendering their study difficult (Kostyrko and Mermod, ). However, they are now attracting increasing attention, notably in oncology, since these “illegitimate” recombination pathways were shown to be more prevalent in tumor cells and to cause chromosomal rearrangements leading to cancer (Bentley et al, ; Simsek et al, ; Tobin et al, ; Zhang and Jasin, ).…”

“…The HR and NHEJ reporter plasmids were kindly provided by V. Gorbunova (University of Rochester, New York) (Mao et al, ). The MMEJ‐specific GFP reporter assay, based on the pGEGFP vector, was constructed as described previously (Kostyrko and Mermod, ). Small interfering RNA duplexes, specifically designed to target the CHO cell homologs of the DNA repair proteins listed in Tables SI and SII, were designed and provided by Microsynth AG (Balgach, Switzerland) (Supplementary Table SIII).…”

POLQ plays a key role in the repair of CRISPR/Cas9‐induced double‐stranded breaks in the moss Physcomitrella patens

“…Alt‐EJ has been described to be a back‐up repair system when other DNA repair pathways are compromised (Wang et al ., ; McVey & Lee, ), but its role could be more pre‐eminent than previously reported (Sfeir & Symington, ; Wang & Xu, ). Different publications have identified POLQ as one of the main enzymes involved in the Alt‐EJ‐mediated DNA repair (Chan et al ., ; Kent et al ., ; Kostyrko & Mermod, ) but its role is still not fully understood. In this study, we analysed the role of the P. patens POLQ protein in DNA repair.…”