SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.     

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

1. Download : Download high-res image (240KB)
2. Download : Download full-size image

     

Regulation of Rad51 function by phosphorylation

Rad51 is a key enzyme involved in DNA double-strand break repair by homologous recombination. Here, we show that in response to DNA damage, budding yeast Rad51 is phosphorylated on Ser 192 in a manner that is primarily mediated by the DNA-damage-responsive protein kinase Mec1. We show that mutating Rad51 Ser 192 to Ala or Glu confers hypersensitivity to DNA damage and homologous-recombination defects. Furthermore, biochemical analyses indicate that Ser 192 is required for Rad51 adenosine triphosphate hydrolysis and DNA-binding activity in vitro, whereas mutation of Ser 192 does not interfere with Rad51 multimer formation. These data suggest a model in which Mec1-mediated phosphorylation of Rad51 Ser 192 in response to DNA damage controls Rad51 activity and DNA repair by homologous recombination.     

Cellular localization of human Rad51C and regulation of ubiquitin-mediated proteolysis of Rad51

Rad51-catalyzed homologous recombination is an important pathway for repair of DNA double strand breaks and maintenance of genome integrity in vertebrate cells. Five proteins referred to as Rad51 paralogs promote Rad51 activity and are proposed to act at various, and in some cases, multiple stages in the recombination pathway. Imaging studies of native Rad51 have revealed its cellular response to DNA damage, yet visualization of the paralog proteins has met with limited success. In this study, we are able to detect endogenous Rad51C and Xrcc3 in human cells. In an effort to determine how Rad51, Rad51C, and Xrcc3 influence the pattern of localization of each other over the time course of DNA damage and repair, we have made the unexpected observation that Rad51 degradation via the ubiquitin-mediated proteasome pathway occurs as a natural part of recombinational DNA repair. Additionally, we find that Rad51C plays an important role in regulating this process. This article contains supplementary material, which may be viewed at the Journal of Cellular Biochemistry website at http://www.interscience.wiley.com/jpages/0730-2312/suppmat/index.html.     

Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination

Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.     

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.     

From yeast to mammals: Recent advances in genetic control of homologous recombination

Misregulation of DNA repair is associated with genetic instability and tumorigenesis. To preserve the integrity of the genome, eukaryotic cells have evolved extremely intricate mechanisms for repairing DNA damage. One type of DNA lesion is a double-strand break (DSB), which is highly toxic when unrepaired. Repair of DSBs can occur through multiple mechanisms. Aside from religating the DNA ends, a homologous template can be used for repair in a process called homologous recombination (HR). One key step in committing to HR is the formation of Rad51 filaments, which perform the homology search and strand invasion steps. In S. cerevisiae, Srs2 is a key regulator of Rad51 filament formation and disassembly. In this review, we highlight potential candidates of Srs2 orthologues in human cells, and we discuss recent advances in understanding how Srs2's so-called “anti-recombinase” activity is regulated.     

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells

1. Download high-res image (217KB)
2. Download full-size image

     

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.     

Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing

Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.     

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein

1. Download : Download high-res image (259KB)
2. Download : Download full-size image

     

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.     

Centromeres as universal hotspots of DNA breakage,driving RAD51-mediated recombination during quiescence

1. Download : Download high-res image (192KB)
2. Download : Download full-size image

     

A PARylation-phosphorylation cascade promotes TOPBP1 loading and RPA-RAD51 exchange in homologous recombination

1. Download : Download high-res image (182KB)
2. Download : Download full-size image

     

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.