S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca(2+), a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.

Determination of the number of RAD51 molecules in different human cell lines

“…Therefore, we treated cells with a cytotoxic agent or ionizing radiation to induce both DNA single-and double-strand breaks. The applied amounts of the chemical bleomycin (12.5 µg/ml) and γ-irradiation (5 Gy) are sufficient to generate several DNA lesions per cell but allow cells to survive due to DNA repair as shown previously [11,23]. In the experiments presented here, we demonstrate that the induction of DNA damage by bleomycin has no influence on the total expression levels of human RAD51 protein when analyzed whole cell extracts (Figure 2 (a)) which is in agreement with a previous study [24].…”

“…Moreover, we determined the RAD51 number in two primary human fibroblast cell lines derived from skin (BJ) and lung (Wi38) [18]. For estimation of endogenous number of RAD51 we used purified RAD51 to prepare standard curves (Figure 1(a)) [11]. From these quantitative western blots, we calculated that approx.…”

“…Thereby, content of RAD51 containing repair foci is comparable to controls after this time[11,25]. For perform its functions RAD51 has to localize on chromatin.…”

“…Subsequently, the resulting nucleoprotein filament is able to invade in homologous sequences and induce recombination processes [4]. The function of RAD51 is analyzed by several biochemical, cell biological, and biophotonic approaches using purified RAD51 protein as well as plasmids expressing RAD51 [5][6][7][8][9][10][11][12][13][14].…”

FLIM‐FRET‐based analysis of S100A11/annexin interactions in living cells

“…As an example, S100A11 is involved in the regulation of actin cytoskeleton in a Ca 2+ ‐dependent manner [ 22 ]. Furthermore, S100A11 seems to be involved in DNA damage repair as it regulates the localization and persistence of its interaction partners RAD54B and RAD51 in DNA repair foci [ 23 , 24 ]. As S100A11 is a binding partner of the receptor for advanced glycation end products (RAGE), S100A11 is supposed to participate in inflammatory response [ 25 ].…”