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bioRxiv · 10.1101/2022.02.23.481227

Biochemical characterization of RAD52-mediated D-loop formation using fluorophore-labeled DNA substrates

Kamoi, K.·Saotome, M.·Kinoshita, C.·Tsuchiya, R.·Kagawa, W.
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Abstract

The human RAD52 protein is thought to have multiple roles in the mechanisms of repairing DNA double-strand breaks that are caused by replication errors and reactive oxygen species. One such role is to mediate the formation of a displacement loop (D-loop), which is a critical reaction intermediate in homologous recombinational repair. RAD52 is suggested to promote the formation of D-loops when facilitating DNA synthesis at stalled or collapsed replication forks during mitosis. However, RAD52-mediated D-loop formation remains poorly characterized, and the detailed molecular mechanism of the D-loop formation reaction catalyzed by RAD52 is still unclear. In the present study, we developed a gel-based assay that enables rapid detection of RAD52-mediated D-loop formation. This assay utilizes a fluorophore-labeled, single-stranded DNA substrate. In addition to the rapid detection of D-loops, D-loop extension was observed when DNA polymerase was added to the reaction. This assay can also be used for screening large numbers of compounds that either stimulate or inhibit RAD52-mediated D-loop formation. The D-loop formation assay developed in this study is potentially useful for mechanistic studies of DSB repair involving RAD52-mediated D-loop formation, as well as for screening compounds with potential therapeutic effects.

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Kamoi, K., Saotome, M., Kinoshita, C., Tsuchiya, R., Kagawa, W.. 2022-02-23. Biochemical characterization of RAD52-mediated D-loop formation using fluorophore-labeled DNA substrates. https://doi.org/10.1101/2022.02.23.481227

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