Figure 4
Two hybrid results. DBD fusions are fusions of the protein listed to the DNA-binding domain of the yeast Gal4 protein, and the AD fusions to the activation domain of Gal4. The X-gal results are from strain Y190, and the ability to grow on media lacking adenine (SC-ade) and the quantitative β-galactosidase activity are from strain PJ69-4A and a rad51∆ derivative of this strain (see Materials and Methods).
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The yeast and human RAD51 genes encode strandtransfer proteins that are thought to be involved in both recombinational repair of DNA damage and meiotic
recombination. In yeast, the Rad51 family of related proteins also includes Rad55, Rad57 and Dmc1. In mammalian cells, five
genes in this family have been identified (HsRAD51, XRCC2, XRCC3, RAD51B/h...
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... The knowledge gained on the specific genes related to ovarian cancer has increased drastically within the last thirty years, starting with the discovery of the well-known BRCA genes in the 1990s (Hall et al., 1990;Wooster et al., 1994). Since then, at least 18 other genes have been identified to be associated with increased risk of ovarian cancer (MLH1 (Lindblom et al., 1993), MSH2 (Peltomäki et al., 1993); PMS2 (Nicolaides et al., 1994), MSH6 (Drummond et al., 1995), EPCAM (Herlyn et al., 1979), TP53 (Lane and Crawford, 1979), CDH1 (Mansouri et al., 1988), MRE11 (Petrini et al., 1995), ATM (Savitsky et al., 1995), RAD50 (Dolganov et al., 1996), BARD1 (Wu et al., 1996), PTEN (Li et al., 1997), RAD51C (Dosanjh et al., 1998), RAD51D (Pittman et al., 1998), NBN (Matsuura et al., 1998), CHEK2 (Bell et al., 1999), BRIP1 (Cantor et al., 2001) and PALB2 (Xia et al., 2006). How strongly these genes are associated with ovarian cancer risk is still discussed. ...
... Several other genes predisposing to ovarian cancer have been identified, including; TP53 (Lane and Crawford, 1979) and CDH1 (Mansouri et al., 1988and 1988, respectively, and MRE11 (Petrini et al., 1995; ATM (Savitsky et al., 1995); RAD50 (Dolganov et al., 1996); BARD1 (Wu et al., 1996); PTEN (Li et al., 1997); RAD51C (Dosanjh et al., 1998); RAD51D (Pittman et al., 1998); NBN (Matsuura et al., 1998); CHEK2 (Bell et al., 1999); BRIP1 (Cantor et al., 2001) and PALB2 (Xia et al., 2006) from 1995 until 2006 (Fig. 1). These susceptibility genes are the most well described in the literature with the highest risk of ovarian cancer. ...
The risk of ovarian, tubal, and peritoneal cancer is related to germline pathogenic variants, and over time, the number of known disease-associated genes has increased significantly. This study reviews the literature regarding the topic from a historical perspective. The aim is to present a timeline of the knowledge gained from the early 1900s until today. The findings are put into perspective by looking at the current gene panel used for screening for suspected hereditary ovarian cancer in Denmark compared to what is known internationally. In 1929, the first familial ovarian cancer incidents were registered, and in 1950, the involvement of a genetic component was suggested for the first time. During the 1970s, several studies reported an accumulation of ovarian cancer in certain families, and during this time, it was discovered that ovarian cancer was linked to both breast cancer and colorectal cancer. The inheritance of cancer disposition has been thoroughly investigated, leading to the discovery of the BRCA genes in the 1990s. Furthermore, new studies based on new genetic technologies have revealed several genes with germline pathogenic variants that increase the risk of ovarian cancer. The identification of these pathogenic variants has led to preventive measures and specific treatment of women with genetic disposition to ovarian cancer. In Denmark, consensus is to include at least ten genes in the screening panel for hereditary ovarian cancer, and in the future additional genes will probably be added.
... DMC1, RAD55, RAD57, SHU1, CSM2 and PSY3 175,[178][179][180] ; whereas vertebrates have seven: DMC1, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and SWSAP1 [181][182][183][184][185][186] . ...
Meiosis, a specialized cell division of the germ cells, is the chromosomal foundation of sexual reproduction in eukaryote organisms. At the outset of meiosis, a programmed induction of double-strand breaks (DSBs) initiates an intricate and highly regulated process for their repair via homologous recombination (HR), leading to the formation of crossovers - reciprocal exchanges of extensive chromosomal segments. The first steps of DSB repair involves the processing of the DSBs and the formation of recombinase-DNA nucleofilaments which, with the help of multiple cofactors, catalyse the search for and invasion of an homologous template. In Arabidopsis thaliana somatic tissues, RAD51 is the recombinase in charge. During meiosis, however, RAD51 plays a supporting role of a meiotic-specific recombinase, DMC1, which catalyses these first steps of HR. In accordance with this, some of the RAD51 instrumental cofactors in somatic repair, such as RAD54 and the RAD51 paralogues RAD51B, RAD51D and XRCC2, are dispensable during meiosis. Given that in absence of DMC1, RAD51 is able to catalyse homology search and strand exchange, proficiently repairing meiotic DSBs, we wondered whether these cofactors become necessary under the hypothesis that their lack of role in wild-type meiosis would be a consequence of the non-catalytic role of RAD51. Indeed, this was the case for RAD54, which we show becomes for meiotic DSB repair and RAD51 nucleofilament function in absence of DMC1. Surprisingly, this is not so for RAD51B, RAD51D and XRCC2, which remained dispensable in the absence of DMC1, hinting at different needs for RAD51-mediated DSB repair in somatic and meiotic cells and leaving the door open for further exploration. In Arabidopsis thaliana, DSBs largely outnumber crossovers ( 20:1). This excess of breaks is repaired using different HR pathways. However, the molecular detection of the non-crossover repair products has been proven challenging, presumably due to the short length of their gene conversion tracts and their sparse localization along the genome. Some of the mechanistic features of both crossover and non-crossover products described in other model organisms, as well as factors that modulate their localization and regulation, remain poorly understood in the model plant. To overcome current technical limitations, we propose the introduction of targeted DSBs at early meiosis using CRISPR/Cas9 systems. By doing so, we would generate an initiation point for HR events at desired locations that would facilitate the detection and mechanistic analysis of repair products. The introduction of DSBs in varied genetic and epigenetic contexts and in multiple genetic backgrounds would permit the direct comparison between controlled conditions to study how they affect DSB repair during meiosis. We designed multiple CRISPR/Cas9 constructs that were able to cleave their target sites at moderate efficiencies and, for some of them, to induce increases of the recombination rate of intervals spanning these sites in individual plants, with high overall variability. We, however, could not detect CRISPR/Cas9-induced DSB repair outcomes at single- molecule level via next-generation sequencing of meiotic products. The use of nucleotide analogues have permitted for decades the cytological and molecular detection of DNA synthesis events, notably DNA replication, in multiple organisms. Given that DNA synthesis is inherent to all meiotic recombination pathways, we wondered if we could profit from one of these analogues (EdU) to label meiotic DSB repair events happening during meiosis in Arabidopsis thaliana. To do so, we designed and validated a protocol that permitted the cytological identification and characterisation of SPO11-dependent meiotic DSB repair-associated DNA synthesis tracts, which offers a new standard tool for the study of meiotic recombination in Arabidopsis. (...)
... At the DNA break, a 3′-single-stranded (ss)DNA overhang is generated and protected by the ssDNA-binding protein RPA (Symington, 2014;Daley et al., 2015). RPA is replaced by the RAD51 recombinase, a rate-limiting step in the HR reaction that is facilitated by multiple recombination mediators (Sung, 1997a;Sung, 1997b;Dosanjh et al., 1998;Sung et al., 2003;Zhao et al., 2015;Belan et al., 2021;Roy et al., 2021). The RAD51-ssDNA nucleoprotein filament then catalyzes the capture of the DNA template and initiates the formation of a displacement loop (D-loop) with the assistance of several RAD51-associated proteins (Petukhova et al., 1998;Tanaka et al., 2000;Miyagawa et al., 2002;Modesti et al., 2007;Wiese et al., 2007;Zhao et al., 2017). ...
Homologous recombination DNA repair (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory HR sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54L, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that concomitant deletion of RAD51AP1 and RAD54L further sensitizes human cancer cell lines to treatment with olaparib, a Poly (adenosine 5′-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which induces DNA replication stress. We also show that the RAD54L paralog RAD54B compensates for RAD54L deficiency, although, surprisingly, less extensively than RAD51AP1. These results, for the first time, delineate RAD51AP1- and RAD54L-dependent sub-pathways and will guide the development of inhibitors that target HR stimulators of strand invasion.
... One of the most interesting genes was a second rad51, rad51C. In humans, this gene is one of five rad51 gene paralogues (Dosanjh et al., 1998). Human rad51 shares homology with the Escherichia coli recA and yeast Saccharomyces cerevisiae rad51 genes, both of which are involved in the repair of double-strand breaks (DSBs) occurring in mitosis and meiosis (Benson et al., 1994;Shinohara et al., 1992). ...
Triploid Nodipecten subnodosus scallops, unlike other mollusks, are completely sterile. In this study we focused on understanding the underlying molecular changes of triploid sterility using a transcriptomic approach. Total RNA from the gonad of diploid scallops in inactive and initial gametogenic stages and triploid scallops of the same cohort and ages were sequenced employing Illumina RNA-Seq. From 68,244 assembled and annotated transcripts, 1120 had terms associated with meiosis checkpoint or arrest, DNA damage response, or recombination. Differential gene expression analyses were conducted by contrasting initial vs. inactive stages of gametogenesis in each ploidy. In diploids, genes participating in homologous recombination during meiosis (msh5 and kdm8), spindle organization (nup62), centrosome formation (cenp-T), and sex differentiation (Ns-dmta2 and pum3), were up-regulated during initial gametogenesis. In triploids, a different set of genes were up-regulated during initial gametogenesis, and included genes involved in the DNA damage response and double strand break repair (rad51-C, xpc, myoVI), in the transition of metaphase/anaphase of mitosis (slp1 and nuf2), as well as genes that trigger both the intrinsic and extrinsic (caspase-3, icad, bmcc1) and extrinsic apoptosis pathways only (tnfr1, dab2ip). The results suggest significant DNA damage in triploids initial gametogenesis, possibly as a consequence of failing to repair double-strand breaks during DNA replication. This coincides with previous observations in which few triploid scallops showed gametic stages more advanced than oogonia or spermatogonia, and when present they were few.
... RAD51C [RAD51 homolog C], is a member of the RAD51 gene family, located on chromosome 17q23, which is expressed with the highest level in testis, followed by the heart muscle, spleen, and prostate, and other various human tissue and organs that encodes strand-transfer proteins in various human tissues. [24,25] BRCA1, BRCA2, PALB2, BRIP1, and RAD51C are involved in DNA damage repair by homologous recombination pathway. [26] For women at the age of 80, there is a 10% risk of developing ovarian cancer carrying a RAD51D mutation. ...
... RAD51C [RAD51 homolog C], is a member of the RAD51 gene family, located on chromosome 17q23, which is expressed with the highest level in testis, followed by the heart muscle, spleen, and prostate, and other various human tissue and organs that encodes strand-transfer proteins in various human tissues. [24,25] BRCA1, BRCA2, PALB2, BRIP1, and RAD51C are involved in DNA damage repair by homologous recombination pathway. [26] For women at the age of 80, there is a 10% risk of developing ovarian cancer carrying a RAD51D mutation. ...
Gynecological cancers are one of the most lethal and deadliest cancers in the world. In India, the prevalence of ovarian cancer accounts for 2.5% to 3%. Despite the availability of improved treatment option along with improved technology, the survival rate of ovarian cancer in the early-stage and the advanced stage is poor. Therefore, due to the heterogeneity of ovarian cancer, to detect it at an early stage and to prevent further mortality turns out to be a big challenge. Researchers are still in the process to identify any single biomarker with good sensitivity and specificity. Various traditional and serum approaches to identify ovarian cancer have been successful in the early stages. The invention of molecular biomarkers such as the use of genomic profiling, DNA methylation, and other approaches have proven to be of higher sensitivity and specificity, which overall affects the prognosis of ovarian cancer. With the use of whole-genome analysis, the detection of possible location of critical tumor suppressor gene (TSGs) in the paired region of chromosomes has been identified, which are associated with BRCA1 and BRCA2 which further makes these novel molecular biomarkers as potential biomarkers. Moreover, studies are required to assess the combined use of traditional, molecular biomarkers that might be useful for enhanced sensitivity and specificity for early detection and prevention of ovarian cancer in early stages which will lead to reduced mortality and good prognosis.
... This overhang is quickly protected by the ssDNA-binding protein RPA. For the HR reaction to continue, RPA must be replaced by the RAD51 recombinase, a rate-limiting process that is dependent on the action of multiple HR mediators (Dosanjh et al., 1998;Godin et al., 2016;Sung, 1997;Sung et al., 2003;Zelensky et al., 2014;Zhao et al., 2015). Then, the RAD51-ssDNA nucleoprotein filament, also known as the presynaptic filament, captures the duplex DNA and generates a displacement loop (D-loop) upon location of the homologous DNA target sequence. ...
NUCKS1 (nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated, vertebrate-specific, and multifunctional protein with a role in DNA damage signaling and repair. Previously, we have shown that NUCKS1 helps maintain homologous recombination (HR) DNA repair in human cells and functions as a tumor suppressor in mice. However, the mechanisms by which NUCKS1 positively impacts these processes had remained unclear. Here, we show that NUCKS1 physically and functionally interacts with the DNA motor protein RAD54. Upon exposure of human cells to DNA-damaging agents, NUCKS1 controls the resolution of RAD54 foci. In unperturbed cells, NUCKS1 prevents RAD54's inappropriate engagement with RAD51AP1. In vitro, NUCKS1 stimulates the ATPase activity of RAD54 and the RAD51-RAD54-mediated strand invasion step during displacement loop formation. Taken together, our data demonstrate that the NUCKS1 protein is an important new regulator of the spatiotemporal events in HR.
... Rad51 has a widely conserved function from yeast to humans [21]. Sharing about 20% to 30% amino acid sequence similarity with Rad51, seven Rad51-like proteins have been identified in vertebrates, namely, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, and XRCC3 [21][22][23][24][25][26][27]. Through co-immunoprecipitation and yeast, two hybrid studies, physical interactions between RAD51 paralogs are detected as two major complexes: RAD51C-XRCC3 (CX3) and RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) [28][29][30]. ...
Radiation sensitive 51 (RAD51) recombinases play crucial roles in meiotic double-strand break (DSB) repair mediated by homologous recombination (HR) to ensure the correct segregation of homologous chromosomes. In this study, we identified the meiotic functions of ZmRAD51C, the maize homolog of Arabidopsis and rice RAD51C. The Zmrad51c mutants exhibited regular vegetative growth but complete sterility for both male and female inflorescence. However, the mutants showed hypersensitivity to DNA damage by mitomycin C. Cytological analysis indicated that homologous chromosome pairing and synapsis were rigorously inhibited, and meiotic chromosomes were often entangled from diplotene to metaphase I, leading to chromosome fragmentation at anaphase I. Immunofluorescence analysis showed that although the signals of the axial element absence of first division (AFD1) and asynaptic1 (ASY1) were normal, the assembly of the central element zipper1 (ZYP1) was severely disrupted. The DSB formation was normal in Zmrad51c meiocytes, symbolized by the regular occurrence of γH2AX signals. However, RAD51 and disrupted meiotic cDNA 1 (DMC1) signals were never detected at the early stage of prophase I in the mutant. Taken together, our results indicate that ZmRAD51C functions crucially for both meiotic DSB repair and homologous recombination in maize.
... In human cells and vertebrates in general, besides RAD51 and DMC1, six RAD51 paralogs have been identified. RAD51B, RAD51C and RAD51D were discovered based on DNA sequence alignments, and XRCC2 and XRCC3 through functional complementation of the ionizing radiation (IR) sensitivity of Chinese hamster mutant cells [14][15][16][17][18]. These five RAD51 paralogs (herein referred to as the classical RAD51 paralogs and focus of this study) are believed to form two functionally distinct heterotypic complexes: the RAD51B-RAD51-C-RAD51D-XRCC2 complex with sub-complexes RAD51B-RAD51C and RAD51D-XRCC2; and the RAD51C-XRCC3 complex ( Fig 1A) [8,9,13,[19][20][21][22][23][24][25]. ...
... In human cells and vertebrates in general, besides RAD51 and DMC1, six RAD51 paralogs have been identified. RAD51B, RAD51C and RAD51D were discovered based on DNA sequence alignments, and XRCC2 and XRCC3 through functional complementation of the ionizing radiation (IR) sensitivity of Chinese hamster mutant cells [14][15][16][17][18]. These five RAD51 paralogs (herein referred to as the classical RAD51 paralogs and focus of this study) are believed to form two functionally distinct heterotypic complexes: the RAD51B-RAD51-C-RAD51D-XRCC2 complex with sub-complexes RAD51B-RAD51C and RAD51D-XRCC2; and the RAD51C-XRCC3 complex ( Fig 1A) [8,9,13,[19][20][21][22][23][24][25]. ...
Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.
