Cellular Redistribution of Rad51 in Response to DNA Damage

Abstract

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damageinduced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damageinduced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.Cellular surveillance of genome integrity and repair of DNA damage are essential processes that ensure proper development and survival of all organisms. DNA double-strand breaks (DSBs) 2 are a particularly deleterious form of genome damage and occur following exposure of cells to exogenous mutagens as well as during normal metabolic processes, e.g. antigen receptor gene rearrangement, restart of stalled replication forks, formation of meiotic DNA crossovers, etc. (1, 2). Mammalian cells use two distinct mechanisms for repair of DSBs, non-homologous end joining and homologous recombination (HR). Processes requiring imprecise DNA repair, such as the creation of antibody diversity, exploit the error-prone non-homologous end joining mechanism. In contrast, HR is an error-free DNA repair pathway and is critical for avoidance of unwanted genetic changes during the meiotic exchange of information between paternal and maternal alleles and for error-free repair of broken chromosomes (3). Rad51 is the central enzymatic component of HR. Upon its regulated recruitment to sites of DNA breaks, Rad51 forms a nucleoprotein filament by polymerizing onto single-stranded DNA at the processed break. This filament catalyzes DNA strand exchange with an undamaged sister chromatid or homologous chromosome, which serves as a template for the restoration of missing genetic information (3, 4).Visible nuclear Rad51 clusters, or foci, form during S-phase and appear to localize to sites of replicating DNA (5-7). A dramatic increase in the number and size of nuclear Rad51 foci is a hallmark of the early cellular response to genomic insult (7-10). The appearance of DNA damage-induced nuclear Rad51 foci is blocked in cells with deficiencies in several HR-related proteins, including Brca2 ...

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair

“…Furthermore, nuclear RAD51 levels in BRCA2-deficient cells were further reduced when RAD51C was depleted (56). These data support the critical role of RAD51C in RAD51 regulation.…”

“…BRCA1 has been implicated as a scaffold protein providing a platform for assembly of various cellular proteins that regulate DNA damage signaling and repair. Notably, RAD51C possesses nuclear localization signal and has been shown to mobilize RAD51 from cytoplasm to nucleus in response to DNA damage (56). The RAD51C R366Q is a mutation in the NLS motif and shows partial loss of function in our assays.…”

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

“…From a wider perspective, our findings of the 53BP1-NUP153/importin-b pathway as an important aspect of the DDR network add to an emerging evidence of subcellular trafficking as an integral part of genome surveillance. Such evidence encompasses ATM-regulated nuclear export 29 and import, 30 as well as the import of essential genome caretakers, such as the FANCD2, RAD51 or BRCA1 repair proteins, [31][32][33] ribonucleotide reductases, 34 DNA damageregulated transcription factors, 35 the ATM-activator protein Aven 36 or p53 tumor suppressor. 37 Although our overall understanding of the role(s) for mammalian NUPs and NPCs in genome maintenance is still in its infancy, significant advances in this research area have already been made based on experiments with yeast models.…”

Posttranslational Modifications of Rad51 Protein and Its Direct Partners: Role and Effect on Homologous Recombination – Mediated DNA Repair

“…Several studies have demonstrated that the overexpression of Rad51 results in its cytoplasmic accumulation (Mladenov et al, 2006) but genotoxic stress triggers the translocation of Rad51 from the cytoplasm to the nucleus (Gildemeister et al, 2009). Plo and colleagues have reported that the nuclear translocation of Rad51 was impaired by AKT1 which repressed HR (Plo et al, 2008).…”

“…Since Rad51 does not have a Nuclear Localisation Signal (NLS) sequence, its nuclear entry likely requires the interaction with other proteins containing functional NLS sequences (Gildemeister et al, 2009). BRCA1 and BRCA2 proteins have both been described as primordial recombination mediators for the nuclear translocation of Rad51.…”