Single molecule microscopy reveals key physical features of repair foci in living cells

Abstract

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions

“…However, it is unlikely that Cdc7 facilitates Rad51 binding to the insoluble fraction by generating a local accumulation of ssDNA because the DNA binding activity of Rad51 is dispensable for its binding to this fraction. Alternatively, MCM phosphorylation might facilitate the formation of a nucleoprotein scaffold or phase-separated liquid compartment as those reported recently at DSB repair centers (Kilic et al, 2019;Miné -Hattab et al, 2021) Functional role of the MCM/Rad51 interaction in replication fork advance and ssDNA repair A remarkable finding of this work is the cell-cycle kinetics of Rad51 and Rad52 binding to the nuclear scaffold: they accumulate in G1 and are released during the S phase, even though HR is inactive in G1 and active in the S phase (Heyer et al, 2010). This kinetics parallels that of the helicase MCM (Aparicio et al, 1997;Liang and Stillman, 1997;Tanaka et al, 1997); indeed, MCM, Rad51, and Rad52 also display similar patterns of binding to this scaffold in the presence of MMS, remaining bound to damaged DNA (Figures 3A, 3B, and S3A).…”

The solid and liquid states of chromatin

“…The principal competing model is that the concentration of chromatin-binding proteins in cells is dictated by differences in the local concentrations of the target chromatin-binding sites [ 92 ]. KMT5C [ 105 , 121 ] and Rad52 [ 122 ] represent the first examples of proteins that behave as expected for proteins that diffuse within chromatin-associated compartments, heterochromatin and DNA double-strand breaks, respectively, but do not freely diffuse across the boundary between the compartment and the nucleoplasm.…”

New Methodologies to Study DNA Repair Processes in Space and Time Within Living Cells

“…Ultimately, single-molecule imaging methods allow the behavior of individual repair proteins to be monitored (see below section “Single-Molecule Approaches to Assess Protein Turnover at Sites of Damage” for more details). They remain, however, difficult to use for non-experts and therefore have not yet been applied extensively in the DNA repair field despite having the potential to provide highly valuable information about protein dynamics ( Miné-Hattab et al, 2021 ).…”

“…Besides the analysis of these search mechanisms, SMLM approaches also allow the behaviour of the repair proteins to be followed at later stages of the DDR in order to investigate how the local environment established nearby the DNA lesions influences the dynamic behavior of the repair factors. In a recent report, Miné-Hattab et al analyzed the individual trajectories of Rad52 (the functional analog of human BRCA2) and RFA1 (a member of the RPA complex) in Saccharomyces cerevisiae ( Miné-Hattab et al, 2021 ). They demonstrate that RFA1 displays subdiffusive motions similar to those reported for the break itself, while Rad52 shows Brownian motion within the repair foci.…”