The RAD51 paralog paradox: Essential functions in DNA repair depend on how proteins move and organize within the dynamic nuclear environment

Abstract Homologous recombination (HR) is complex multi-step genome transaction essential to support replication and repair DNA breaks. The defining step of HR is catalysed in mammalian cells by the RAD51 protein. It is well defined that a RAD51 filament assembled on single stranded DNA can catalyse DNA strand exchange in vitro.

In vivo, both the efficiency and control of this process are achieved in cooperation with a host of partner proteins. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are among the mediator proteins with critical roles in HR. They have sequence similarity to RAD51 but do not themselves catalyse DNA strand exchange.

RAD51 paralogs are believed to function in two major complexes, BCDX2 (RAD51B, C, D and XRCC2) and the CX3 (RAD51C and XRCC3).

The biochemical activity of the paralogs, based on studies with subsets of purified components, suggest they affect RAD51 filament formation and dynamics. However, their relatively subtle biochemical activities do not explain their in vivo importance.

We aim to identify the essential in vivo functions of the RAD51 paralogs during homology-directed DNA double strand break repair by determining their dynamic nuclear organization in response to DNA damage.

We have developed a toolbox of methods including cell engineering, dynamic super-resolution imaging and image analysis algorithms to analyse HR proteins in vivo.

We will create endogenous-expressed fluorescent-tagged RAD51 paralogs and follow their function in the complex environment of the nucleus.

We will focus first on RAD51C (representing both complexes), then RAD51B, and XRCC3 (respectively representing distinct complexes).

We will quantitatively describe the diffusive behaviour in living cells and determine their nanoscale localization at sites of DNA damage.

By degron tags we will induce controlled removal of a specific paralog and determine the influence on diffusive behaviour and nanoscale organization of other HR proteins such as RAD51 and BRCA2. This will define essential parameters of HR function and control. We hope to solve the RAD51 paralog paradox by identifying critical in vivo activities.