The protein phosphatase EYA4 promotes homologous recombination (HR) through dephosphorylation of tyrosine 315 on RAD51.

Efficient DNA repair and limitation of genome rearrangements rely on crosstalk between different DNA double-strand break (DSB) repair pathways, and their synchronization with the cell cycle. The selection, timing and efficacy of DSB repair pathways are influenced by post-translational modifications of histones and DNA damage repair (DDR) proteins, such as phosphorylation. While the importance of kinases and serine/threonine phosphatases in DDR have been extensively studied, the role of tyrosine phosphatases in DNA repair remains poorly understood. In this study, we have identified EYA4 as the protein phosphatase that dephosphorylates RAD51 on residue Tyr315. Through its Tyr phosphatase activity, EYA4 regulates RAD51 localization, presynaptic filament formation, foci formation, and activity. Thus, it is essential for homologous recombination (HR) at DSBs. DNA binding stimulates EYA4 phosphatase activity. Depletion of EYA4 decreases single-stranded DNA accumulation following DNA damage and impairs HR, while overexpression of EYA4 in cells promotes dephosphorylation and stabilization of RAD51, and thereby nucleoprotein filament formation. Our data have implications for a pathological version of RAD51 in EYA4-overexpressing cancers.

[Peracute Diseases of the Esophagus - Bleeding from Esophageal Varices, Esophageal Varices]. / Perakute Erkrankungen des Ösophagus.

Peracute Diseases of the Esophagus - Bleeding from Esophageal Varices, Esophageal Varices Abstract. Due to a permanently increased portal venous pressure - usually due to infectious or ethyltoxic liver cirrhosis - varices can form in the lower esophagus due to expansion of the submucosal venous plexus. Acute bleeding from the esophageal varices is a life-threatening situation. In therapy, a distinction is made between primary prophylaxis of bleeding, control of acute bleeding and prevention of recurrent bleeding. In addition to non-selective betablockers, the transjugular intrahepatic portosystemic shunt (TIPS), which is introduced radiologically, plays a decisive role today, especially in the prophylaxis of recurrent bleeding. Apart from special indications, surgical shunt procedures are only of historical value. In liver cirrhosis patients, liver transplantation represents a causal treatment and lasting cure for esophageal varices.

A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution.

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.

hSSB1 associates with and promotes stability of the BLM helicase.

BACKGROUND: Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. RESULTS: In this study, we demonstrate that hSSB1 and BLM helicase form a complex in cells and the interaction is altered in response to ionising radiation (IR). BLM and hSSB1 also co-localised at nuclear foci following IR-induced double strand breaks and stalled replication forks. We show that hSSB1 depleted cells contain less BLM protein and that this deficiency is due to proteasome mediated degradation of BLM. Consequently, there is a defect in recruitment of BLM to chromatin in response to ionising radiation-induced DSBs and to hydroxyurea-induced stalled and collapsed replication forks. CONCLUSIONS: Our data highlights that BLM helicase and hSSB1 function in a dynamic complex in cells and that this complex is likely required for BLM protein stability and function.

hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway.

The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome.

A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation.

Thymine DNA glycosylase (TDG) functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU) paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP) of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs). Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer.

Novel insight into the composition of human single-stranded DNA-binding protein 1 (hSSB1)-containing protein complexes.

BACKGROUND: Single-stranded DNA-binding proteins are essential cellular components required for the protection, metabolism and processing of single-stranded DNA. Human single-stranded DNA-binding protein 1 (hSSB1) is one such protein, with described roles in genome stability maintenance and transcriptional regulation. As yet, however, the mechanisms through which hSSB1 functions and the binding partners with which it interacts remain poorly understood. RESULTS: In this work, hSSB1 was immunoprecipitated from cell lysate samples that had been enriched for non-soluble nuclear proteins and those associating with hSSB1 identified by mass spectrometry. In doing so, 334 potential hSSB1-associating proteins were identified, with known roles in a range of distinct biological processes. Unexpectedly, whilst hSSB1 has largely been studied in a genome stability context, few other DNA repair or replication proteins were detected. By contrast, a large number of proteins were identified with roles in mRNA metabolism, reflecting a currently emerging area of hSSB1 study. In addition, numerous proteins were detected that comprise various chromatin-remodelling complexes. CONCLUSIONS: These findings provide new insight into the binding partners of hSSB1 and will likely function as a platform for future research.

Nucleophosmin: from structure and function to disease development.

Nucleophosmin (NPM1) is a critical cellular protein that has been implicated in a number of pathways including mRNA transport, chromatin remodeling, apoptosis and genome stability. NPM1 function is a critical requirement for normal cellular biology as is underlined in cancer where NPM1 is commonly overexpressed, mutated, rearranged and sporadically deleted. Consistent with a multifunctional role within the cell, NPM1 can function not only as a proto-oncogene but also as a tumor suppressor. The aim of this review is to look at the less well-described role of NPM1 in the DNA repair pathways as well as the role of NPM1 in the regulation of apoptosis and its mutation in cancers.

Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer.

Designer peptides have recently attracted attention as self-assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate-rich peptides in particular have shown potential as salt-resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled-coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate-proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography-mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate-proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C-termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol(-1) , with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol(-1) . We determined an interfacial pressure of 29 mN m(-1) for both intact and cleaved concatemer at the air-water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat-stable emulsions with droplet sizes in the nanometer range.

Néstor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation.

BACKGROUND: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure. RESULTS: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope. CONCLUSIONS: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

Tumor-associated mutations in O6 -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality.

MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O6 -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O6 -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization.

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

Toward Food Sovereignty for Coastal Communities of Eastern Québec: Co-designing A Website to Support Consumption of Edible Resources from the St. Lawrence River, Estuary, and Gulf.

Background. Despite the abundance and proximity of edible marine resources, coastal communities along the St. Lawrence in Eastern Québec rarely consume these resources. Within a community-based food sovereignty project, Manger notre Saint-Laurent ("Sustenance from our St. Lawrence"), members of participating communities (3 non-Indigenous, 1 Indigenous) identified a need for a web-based decision tool to help make informed consumption choices. Methods. We thus aimed to co-design a prototype website that facilitates informed choices about consuming local edible marine resources based on seasonal and regional availability, food safety, nutrition, and sustainability, with community members, regional stakeholders, and experts in user experience design and web development. We conducted 48 interviews with a variety of people over 3 iterative cycles, assessing the prototype's ease of use with a validated measure, the System Usability Scale. Results. Community members, regional stakeholders, and other experts identified problematic elements in initial versions of the website (e.g., confusing symbols). We resolved issues and added features people identified as useful. Usability scores reached "best imaginable" for both the second and the third versions and did not differ significantly between sociodemographic groups. The final prototype includes a tool to explore each species and index cards to regroup accurate evidence relevant to each species. Conclusions. Engaging co-designers with different sociodemographic characteristics brought together a variety of perspectives. Several components would not have been included without co-designers' input; other components were greatly improved thanks to their feedback. Co-design approaches in research and intervention development are preferable to foster the inclusion of a variety of people. Once the prototype is programmed and available online, we hope to evaluate the website to determine its effects on food choices.

Structure and vascular tissue expression of duplicated TERMINAL EAR1-like paralogues in poplar.

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula x P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).

hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases.

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress.

The maintenance of genome stability is an essential cellular process to prevent the development of diseases including cancer. hSSB1 (NABP2/ OBFC2A) is a critical component of the DNA damage response where it participates in the repair of double-strand DNA breaks and in base excision repair of oxidized guanine residues (8-oxoguanine) by aiding the localization of the human 8-oxoguanine glycosylase (hOGG1) to damaged DNA. Here we demonstrate that following oxidative stress, hSSB1 is stabilized as an oligomer which is required for hSSB1 to function in the removal of 8-oxoguanine. Monomeric hSSB1 shows a decreased affinity for oxidized DNA resulting in a cellular 8-oxoguanine-repair defect and in the absence of ATM signaling initiation. While hSSB1 oligomerization is important for the removal of 8-oxoguanine from the genome, it is not required for the repair of double-strand DNA-breaks by homologous recombination. These findings demonstrate a novel hSSB1 regulatory mechanism for the repair of damaged DNA.

Organization and expression of the GSK3/shaggy kinase gene family in the moss Physcomitrella patens suggest early gene multiplication in land plants and an ancestral response to osmotic stress.

GSK3/Shaggy kinases are involved in a wide range of fundamental processes in animal development and metabolism. In angiosperm plants, these kinases are encoded by moderate-sized gene families, which appear to have a complex set of functions. Here, we present the characterization of five members of the GSK3/Shaggy gene family in the bryophyte Physcomitrella patens. The P. patens GSK3/Shaggy kinases (PpSK) are organized in a group of closely related paralogues with respect to their gene sequence and structure. Indeed, a phylogenetic analysis of the GSK3/Shaggy kinase sequences from plants and animals showed that the five PpSK proteins are monophyletic, and closer to subgroups I and IV described in angiosperms. Expression analyses performed by quantitative real-time RT-PCR on a wide range of growing conditions showed that PpSK genes responded only to either desiccation, PEG or sorbitol. As demonstrated by both inductions of marker genes and protonemal cell plasmolyses, these treatments resulted in a hyperosmotic stress. Altogether, these data suggest that (1) GSK3/Shaggy kinase gene multiplication occurred early in plant evolution, before the separation between bryophytes and vascular plants, and (2) both gene loss and duplication occurred in the ancestor of P. patens along with functional gene diversification in angiosperms. However, conservation of the transcriptional responses between Physcomitrella and Arabidopsis suggests the identification of an ancestral response of the GSK3/Shaggy kinases genes to osmotic stress.

Expression patterns of TEL genes in Poaceae suggest a conserved association with cell differentiation.

Poaceae species present a conserved distichous phyllotaxy (leaf position along the stem) and share common properties with respect to leaf initiation. The goal of this work was to determine if these common traits imply common genes. Therefore, homologues of the maize TERMINAL EAR1 gene in Poaceae were studied. This gene encodes an RNA-binding motif (RRM) protein, that is suggested to regulate leaf initiation. Using degenerate primers, one unique tel (terminal ear1-like) gene from seven Poaceae members, covering almost all the phylogenetic tree of the family, was identified by PCR. These genes present a very high degree of similarity, a much conserved exon-intron structure, and the three RRMs and TEL characteristic motifs. The evolution of tel sequences in Poaceae strongly correlates with the known phylogenetic tree of this family. RT-PCR gene expression analyses show conserved tel expression in the shoot apex in all species, suggesting functional orthology between these genes. In addition, in situ hybridization experiments with specific antisense probes show tel transcript accumulation in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells. Tel expression is not restricted to initiating leaves as it is also found in pro-vascular tissues, root meristems, and immature inflorescences. Therefore, these results suggest that TEL is not only associated with leaf initiation but more generally with cell differentiation in Poaceae.