Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Functional Characterization of the Human BRCA1 ∆11 Splicing Isoforms in Yeast.

BRCA1, a crucial tumor suppressor gene, has several splicing isoforms, including Δ9-11, Δ11, and Δ11q, which lack exon 11, coding for significant portions of the protein. These isoforms are naturally present in both normal and cancerous cells, exhibiting altered activity compared to the full-length BRCA1. Despite this, the impact on cancer risk of the germline intronic variants promoting the exclusive expression of these Δ11 isoforms remains uncertain. Consequently, they are classified as variants of uncertain significance (VUS), posing challenges for traditional genetic classification methods due to their rarity and complexity. Our research utilizes a yeast-based functional assay, previously validated for assessing missense BRCA1 variants, to compare the activity of the Δ11 splicing isoforms with known pathogenic missense variants. This approach allows us to elucidate the functional implications of these isoforms and determine whether their exclusive expression could contribute to increased cancer risk. By doing so, we aim to provide insights into the pathogenic potential of intronic VUS-generating BRCA1 splicing isoforms and improve the classification of BRCA1 variants.

The BRCA1 c.4096+1G>A Is a Founder Variant Which Originated in Ancient Times.

Approximately 30-50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45-79 percent and 39-48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago-around 3000 years ago.

RAD52 influences the effect of BRCA1/2 missense variants on homologous recombination and gene reversion in Saccharomyces cerevisiae.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae.

Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.

Inhibition of DNA Repair in Cancer Therapy: Toward a Multi-Target Approach.

Alterations in DNA repair pathways are one of the main drivers of cancer insurgence. Nevertheless, cancer cells are more susceptible to DNA damage than normal cells and they rely on specific functional repair pathways to survive. Thanks to advances in genome sequencing, we now have a better idea of which genes are mutated in specific cancers and this prompted the development of inhibitors targeting DNA repair players involved in pathways essential for cancer cells survival. Currently, the pivotal concept is that combining the inhibition of mechanisms on which cancer cells viability depends is the most promising way to treat tumorigenesis. Numerous inhibitors have been developed and for many of them, efficacy has been demonstrated either alone or in combination with chemo or radiotherapy. In this review, we will analyze the principal pathways involved in cell cycle checkpoint and DNA repair focusing on how their alterations could predispose to cancer, then we will explore the inhibitors developed or in development specifically targeting different proteins involved in each pathway, underscoring the rationale behind their usage and how their combination and/or exploitation as adjuvants to classic therapies could help in patients clinical outcome.

Whole-exome analysis of a Li-Fraumeni family trio with a novel TP53 PRD mutation and anticipation profile.

Li-Fraumeni syndrome is a clinically heterogeneous familial cancer predisposition syndrome with autosomal-dominant inheritance caused by heterozygous germline mutations in the TP53 gene. We here analyze the genetic background of a family with a 4-year-proband presented with a Li-Fraumeni tumor. The mother developed breast cancer at age 37 and the proband died at age 8. We performed Sanger sequencing and whole-exome sequencing on peripheral blood DNA from proband and relatives. Data analysis selected only high-quality score and depth reads, rare variants and protein impact involving missense, non-sense, frameshift and splice disrupt mutations. Disease implicated variants and predicted deleterious alterations were also chosen. TP53 genetic testing revealed a never reported TP53 deletion arose as de novo mutation in the mother and inherited by the proband. We then performed whole-exome analysis of the trio to uncover inherited variants from the father that potentially worsen the already altered genetic background in the proband. No pathogenic variants were inherited in autosomal recessive, de novo dominant or X-linked recessive manner. Comparing proband and father exome we detected 25 predicted deleterious variants including a nonsense mutation in ERCC3. Those inherited mutations are possible candidate modifiers linked to TP53, explaining the proband accelerated tumor onset compared to the mother and providing a possible explanation of the genetic anticipation event in this Li-Fraumeni family.

CRIMEtoYHU: a new web tool to develop yeast-based functional assays for characterizing cancer-associated missense variants.

Evaluation of the functional impact of cancer-associated missense variants is more difficult than for protein-truncating mutations and consequently standard guidelines for the interpretation of sequence variants have been recently proposed. A number of algorithms and software products were developed to predict the impact of cancer-associated missense mutations on protein structure and function. Importantly, direct assessment of the variants using high-throughput functional assays using simple genetic systems can help in speeding up the functional evaluation of newly identified cancer-associated variants. We developed the web tool CRIMEtoYHU (CTY) to help geneticists in the evaluation of the functional impact of cancer-associated missense variants. Humans and the yeast Saccharomyces cerevisiae share thousands of protein-coding genes although they have diverged for a billion years. Therefore, yeast humanization can be helpful in deciphering the functional consequences of human genetic variants found in cancer and give information on the pathogenicity of missense variants. To humanize specific positions within yeast genes, human and yeast genes have to share functional homology. If a mutation in a specific residue is associated with a particular phenotype in humans, a similar substitution in the yeast counterpart may reveal its effect at the organism level. CTY simultaneously finds yeast homologous genes, identifies the corresponding variants and determines the transferability of human variants to yeast counterparts by assigning a reliability score (RS) that may be predictive for the validity of a functional assay. CTY analyzes newly identified mutations or retrieves mutations reported in the COSMIC database, provides information about the functional conservation between yeast and human and shows the mutation distribution in human genes. CTY analyzes also newly found mutations and aborts when no yeast homologue is found. Then, on the basis of the protein domain localization and functional conservation between yeast and human, the selected variants are ranked by the RS. The RS is assigned by an algorithm that computes functional data, type of mutation, chemistry of amino acid substitution and the degree of mutation transferability between human and yeast protein. Mutations giving a positive RS are highly transferable to yeast and, therefore, yeast functional assays will be more predictable. To validate the web application, we have analyzed 8078 cancer-associated variants located in 31 genes that have a yeast homologue. More than 50% of variants are transferable to yeast. Incidentally, 88% of all transferable mutations have a reliability score >0. Moreover, we analyzed by CTY 72 functionally validated missense variants located in yeast genes at positions corresponding to the human cancer-associated variants. All these variants gave a positive RS. To further validate CTY, we analyzed 3949 protein variants (with positive RS) by the predictive algorithm PROVEAN. This analysis shows that yeast-based functional assays will be more predictable for the variants with positive RS. We believe that CTY could be an important resource for the cancer research community by providing information concerning the functional impact of specific mutations, as well as for the design of functional assays useful for decision support in precision medicine.

BRCA1 Circos: a visualisation resource for functional analysis of missense variants.

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.

Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae.

Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3' overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway.

Functional assays for analysis of variants of uncertain significance in BRCA2.

Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore, alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication, and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review, we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including variants of uncertain significance analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk.

Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae.

BACKGROUND: Homologous recombination mediated gene targeting is still too inefficient to be applied extensively in genomics and gene therapy. Although sequence-specific nucleases could greatly stimulate gene targeting efficiency, the off-target cleavage sites of these nucleases highlighted the risk of this strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene knockouts, since several studies indicate that these vectors are able to induce site-specific genome alterations at high frequency. Since each targeted event is accompanied by at least ten random integration events, increasing our knowledge regarding the mechanisms behind these events is necessary in order to understand the potential of AAV-mediated gene targeting for therapy application. Moreover, the role of AAV regulatory proteins (Rep) and inverted terminal repeated sequences (ITRs) in random and homologous integration is not completely known. In this study, we used the yeast Saccharomyces cerevisiae as a genetic model system to evaluate whether the presence of ITRs in the integrating plasmid has an effect on gene targeting and random integration. RESULTS: We have shown that the presence of ITRs flanking a gene targeting vector containing homology to its genomic target decreased the frequency of random integration, leading to an increase in the gene targeting/random integration ratio. On the other hand, the expression of Rep proteins, which produce a nick in the ITR, significantly increased non-homologous integration of a DNA fragment sharing no homology to the genome, but had no effect on gene targeting or random integration when the DNA fragment shared homology with the genome. Molecular analysis showed that ITRs are frequently conserved in the random integrants, and that they induce rearrangements. CONCLUSIONS: Our results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.

Silencing of BRCA2 decreases anoikis and its heterologous expression sensitizes yeast cells to acetic acid-induced programmed cell death.

Adhesion of normal epithelial cells to the extracellular matrix (ECM) is essential for survival. Cell detachment from ECM induces a specific form of programmed cell death (PCD) termed anoikis. BRCA2, a tumor suppressor gene whose mutations confer predisposition to cancer, has been implicated in the regulation of DNA repair, transcription, cell proliferation, and apoptosis. However, the potential role of BRCA2 in the regulation of anoikis has not been investigated. Here, we found that suppression of BRCA2 expression by short hairpin RNA promoted resistance to anoikis in prostate, breast and thyroid normal epithelial cells, which was accompanied by reduced caspases 3/7 levels and activity. Using yeast as a model, we assessed that expression of human BRCA2 does not induce cell death by itself but it can promote acetic acid-induced PCD (AA-PCD). Induction of BRCA2 expression decreased cell survival and increased the number of cells positive to different apoptotic markers, including DNA fragmentation and phosphatidylserine externalization en route to AA-PCD. A higher increase in ROS levels occurred in the early phase of AA-PCD in BRCA2-expressing yeast cells compared with non-expressing cells. Accordingly, a delay in the initial burst of ROS levels was observed in BRCA2-knockdown anoikis-resistant human cells. Treatment with the antioxidants N-acetylcysteine or ascorbic acid reduced sensitivity to anoikis in human cells and inhibited AA-PCD in yeast cells expressing BRCA2. Taken together, these results show a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS production and/or detoxification by BRCA2 during PCD processes.

The expanding role of yeast in cancer research and diagnosis: insights into the function of the oncosuppressors p53 and BRCA1/2.

When the glucose supply is high, despite the presence of oxygen, Saccharomyces cerevisiae uses fermentation as its main metabolic pathway and switches to oxidative metabolism only when this carbon source is limited. There are similarities between glucose-induced repression of oxidative metabolism of yeast and metabolic reprogramming of tumor cells. The glucose-induced repression of oxidative metabolism is regulated by oncogene homologues in yeast, such as RAS and Sch9p, the yeast homologue of Akt. Yeast also undergoes an apoptosis-like programmed cell death process sharing several features with mammalian apoptosis, including oxidative stress and a major role played by mitochondria. Evasion of apoptosis and sustained proliferative signaling are hallmarks of cancer. This, together with the possibility of heterologous expression of human genes in yeast, has allowed new insights to be obtained into the function of mammalian oncogenes/oncosuppressors. Here, we elaborate on the similarities between tumor and yeast cells underpinning the use of this model organism in cancer research. We also review the achievements obtained through heterologous expression in yeast of p53, BRCA1, and BRCA2, which are among the best-known cancer-susceptibility genes, with the aim of understanding their role in tumorigenesis. Yeast-cell-based functional assays for cancer genetic testing will also be dealt with.

Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae.

The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15-30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.

A guide for functional analysis of BRCA1 variants of uncertain significance.

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56-80% for breast cancer and 15-60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.

Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.

BACKGROUND: The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. RESULTS: To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. CONCLUSIONS: Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein.

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a 'protein delivery approach' offers a new tool for developing novel strategies for genome modification and gene therapy applications.