Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (Km = 26 µM) than chloride (Km = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus.

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

Halogenation in Fungi: What Do We Know and What Remains to Be Discovered?

In nature, living organisms produce a wide variety of specialized metabolites to perform many biological functions. Among these specialized metabolites, some carry halogen atoms on their structure, which can modify their chemical characteristics. Research into this type of molecule has focused on how organisms incorporate these atoms into specialized metabolites. Several families of enzymes have been described gathering metalloenzymes, flavoproteins, or S-adenosyl-L-methionine (SAM) enzymes that can incorporate these atoms into different types of chemical structures. However, even though the first halogenation enzyme was discovered in a fungus, this clade is still lagging behind other clades such as bacteria, where many enzymes have been discovered. This review will therefore focus on all halogenation enzymes that have been described in fungi and their associated metabolites by searching for proteins available in databases, but also by using all the available fungal genomes. In the second part of the review, the chemical diversity of halogenated molecules found in fungi will be discussed. This will allow the highlighting of halogenation mechanisms that are still unknown today, therefore, highlighting potentially new unknown halogenation enzymes.

Pentadecaibins I-V: 15-Residue Peptaibols Produced by a Marine-Derived Trichoderma sp. of the Harzianum Clade.

In the course of investigations on peptaibol chemodiversity from marine-derived Trichoderma spp., five new 15-residue peptaibols named pentadecaibins I-V (1-5) were isolated from the solid culture of the strain Trichoderma sp. MMS1255 belonging to the T. harzianum species complex. Phylogenetic analyses allowed precise positioning of the strain close to T. lentiforme lineage inside the Harzianum clade. Peptaibol sequences were elucidated on the basis of their MS/MS fragmentation and extensive 2D NMR experiments. Amino acid configurations were determined by Marfey's analyses. The pentadecaibins are based on the sequences Ac-Aib1-Gly2-Ala3-Leu4-Aib/Iva5-Gln6-Aib/Iva7-Val/Leu8-Aib9-Ala10-Aib11-Aib12-Aib13-Gln14-Pheol15. Characteristic of the pentadecaibin sequences is the lack of the Aib-Pro motif commonly present in peptaibols produced by Trichoderma spp. Genome sequencing of Trichoderma sp. MMS1255 allowed the detection of a 15-module NRPS-encoding gene closely associated with pentadecaibin biosynthesis. Pentadecaibins were assessed for their potential antiproliferative and antimicrobial activities.

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis.

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.

Comparative genomics applied to Mucor species with different lifestyles.

BACKGROUND: Despite a growing number of investigations on early diverging fungi, the corresponding lineages have not been as extensively characterized as Ascomycota or Basidiomycota ones. The Mucor genus, pertaining to one of these lineages is not an exception. To this date, a restricted number of Mucor annotated genomes is publicly available and mainly correspond to the reference species, Mucor circinelloides, and to medically relevant species. However, the Mucor genus is composed of a large number of ubiquitous species as well as few species that have been reported to specifically occur in certain habitats. The present study aimed to expand the range of Mucor genomes available and identify potential genomic imprints of adaptation to different environments and lifestyles in the Mucor genus. RESULTS: In this study, we report four newly sequenced genomes of Mucor isolates collected from non-clinical environments pertaining to species with contrasted lifestyles, namely Mucor fuscus and Mucor lanceolatus, two species used in cheese production (during ripening), Mucor racemosus, a recurrent cheese spoiler sometimes described as an opportunistic animal and human pathogen, and Mucor endophyticus, a plant endophyte. Comparison of these new genomes with those previously available for six Mucor and two Rhizopus (formerly identified as M. racemosus) isolates allowed global structural and functional description such as their TE content, core and species-specific genes and specialized genes. We proposed gene candidates involved in iron metabolism; some of these genes being known to be involved in pathogenicity; and described patterns such as a reduced number of CAZymes in the species used for cheese ripening as well as in the endophytic isolate that might be related to adaptation to different environments and lifestyles within the Mucor genus. CONCLUSIONS: This study extended the descriptive data set for Mucor genomes, pointed out the complexity of obtaining a robust phylogeny even with multiple genes families and allowed identifying contrasting potentially lifestyle-associated gene repertoires. The obtained data will allow investigating further the link between genetic and its biological data, especially in terms of adaptation to a given habitat.

Comparative analysis of five Mucor species transcriptomes.

Mucor species belong to the Mucorales order within the Mucoromycota phylum, an early diverging fungal lineage. Although Mucor species are often ubiquitous some species have been reported to specifically occur in certain ecological niches. In this study, similarities and differences of a representative set of Mucor species with contrasted lifestyles were investigated at the transcriptome level. Five strains pertaining to five different species were studied, namely M. fuscus and M. lanceolatus, two species used in cheese production (during ripening), M. racemosus, a recurrent cheese spoiler sometimes described as an opportunistic pathogen, M. circinelloides, often described as an opportunistic pathogen and M. endophyticus, a plant endophyte. A core transcriptome was delimited and a phylogenetic analysis led to an altered phylogenetic placement of M. endophyticus compared to previously published topologies. Interestingly, the core transcriptome comprising 5566 orthogroups included genes potentially involved in secondary metabolism. As expected, given the wide taxonomic range investigated, the five transcriptomes also displayed specificities that can be, for some of them, linked to the different lifestyles such as differences in the composition of transcripts identified as virulence factors or carbohydrate transporters.

Penicillium roqueforti PR toxin gene cluster characterization.

PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

Identification and quantification of antifungal compounds produced by lactic acid bacteria and propionibacteria.

Fungal growth in bakery products represents the most frequent cause of spoilage and leads to economic losses for industrials and consumers. Bacteria, such as lactic acid bacteria and propionibacteria, are commonly known to play an active role in preservation of fermented food, producing a large range of antifungal metabolites. In a previous study (Le Lay et al., 2016), an extensive screening performed both in vitro and in situ allowed for the selection of bacteria exhibiting an antifungal activity. In the present study, active supernatants against Penicillium corylophilum and Aspergillus niger were analyzed to identify and quantify the antifungal compounds associated with the observed activity. Supernatant treatments (pH neutralization, heating and addition of proteinase K) suggested that organic acids played the most important role in the antifungal activity of each tested supernatant. Different methods (HPLC, mass spectrometry, colorimetric and enzymatic assays) were then applied to analyze the supernatants and it was shown that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels such as phenyllactic, hydroxyphenyllactic, azelaic and caproic acids. Based on these results, various combinations of the identified compounds were used to evaluate their effect on conidial germination and fungal growth of P. corylophilum and Eurotium repens. Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the identified molecules in the antifungal activity. The obtained results suggested that acetic acid was mainly responsible for the antifungal activity against P. corylophilum and played an important role in E. repens inhibition.

Deep Subseafloor Fungi as an Untapped Reservoir of Amphipathic Antimicrobial Compounds.

The evolving global threat of antimicrobial resistance requires a deep renewal of the antibiotic arsenal including the isolation and characterization of new drugs. Underexplored marine ecosystems may represent an untapped reservoir of novel bioactive molecules. Deep-sea fungi isolated from a record-depth sediment core of almost 2000 m below the seafloor were investigated for antimicrobial activities. This antimicrobial screening, using 16 microbial targets, revealed 33% of filamentous fungi synthesizing bioactive compounds with activities against pathogenic bacteria and fungi. Interestingly, occurrence of antimicrobial producing isolates was well correlated with the complexity of the habitat (in term of microbial richness), as higher antimicrobial activities were obtained at specific layers of the sediment core. It clearly highlights complex deep-sea habitats as chemical battlefields where synthesis of numerous bioactive compounds appears critical for microbial competition. The six most promising deep subseafloor fungal isolates were selected for the production and extraction of bioactive compounds. Depending on the fungal isolates, antimicrobial compounds were only biosynthesized in semi-liquid or solid-state conditions as no antimicrobial activities were ever detected using liquid fermentation. An exception was made for one fungal isolate, and the extraction procedure designed to extract amphipathic compounds was successful and highlighted the amphiphilic profile of the bioactive metabolites.

Species richness and adaptation of marine fungi from deep-subseafloor sediments.

The fungal kingdom is replete with unique adaptive capacities that allow fungi to colonize a wide variety of habitats, ranging from marine habitats to freshwater and terrestrial habitats. The diversity, importance, and ecological roles of marine fungi have recently been highlighted in deep-subsurface sediments using molecular methods. Fungi in the deep-marine subsurface may be specifically adapted to life in the deep biosphere, but this can be demonstrated only using culture-based analyses. In this study, we investigated culturable fungal communities from a record-depth sediment core sampled from the Canterbury Basin (New Zealand) with the aim to reveal endemic or ubiquist adapted isolates playing a significant ecological role(s). About 200 filamentous fungi (68%) and yeasts (32%) were isolated. Fungal isolates were affiliated with the phyla Ascomycota and Basidiomycota, including 21 genera. Screening for genes involved in secondary metabolite synthesis also revealed their bioactive compound synthesis potential. Our results provide evidence that deep-subsurface fungal communities are able to survive, adapt, grow, and interact with other microbial communities and highlight that the deep-sediment habitat is another ecological niche for fungi.

Discovery of a novel iota carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora.

Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of carrageenans. As a result of the screening, an iota-carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain Psc(T). It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc ι-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH ~8.3 and at 40 ± 5°C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc ι-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-carrageenan. The purified enzyme could transform iota-carrageenan into hybrid iota-/alpha- or pure alpha-carrageenan under controlled conditions. The gene encoding Psc ι-CgsA, a protein of 1038 amino acids, was cloned into Escherichia coli, and the sequence analysis revealed that Psc ι-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P. carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes.

The Vanadium Iodoperoxidase from the marine flavobacteriaceae species Zobellia galactanivorans reveals novel molecular and evolutionary features of halide specificity in the vanadium haloperoxidase enzyme family.

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.

Structure/function analysis of a type iii polyketide synthase in the brown alga Ectocarpus siliculosus reveals a biochemical pathway in phlorotannin monomer biosynthesis.

Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85-Å resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

A new method to identify flanking sequence tags in chlamydomonas using 3'-RACE.

BACKGROUND: The green alga Chlamydomonas reinhardtii, although a premier model organism in biology, still lacks extensive insertion mutant libraries with well-identified Flanking Sequence Tags (FSTs). Rapid and efficient methods are needed for FST retrieval. RESULTS: Here, we present a novel method to identify FSTs in insertional mutants of Chlamydomonas. Transformants can be obtained with a resistance cassette lacking a 3' untranslated region (UTR), suggesting that the RNA that is produced from the resistance marker terminates in the flanking genome when it encounters a cleavage/polyadenylation signal. We have used a robust 3'-RACE method to specifically amplify such chimeric cDNAs. Out of 38 randomly chosen transformants, 27 (71%) yielded valid FSTs, of which 23 could be unambiguously mapped to the genome. Eighteen of the mutants lie within a predicted gene. All but two of the intragenic insertions occur in the sense orientation with respect to transcription, suggesting a bias against situations of convergent transcription. Among the 14 insertion sites tested by genomic PCR, 12 could be confirmed. Among these are insertions in genes coding for PSBS3 (possibly involved in non-photochemical quenching), the NimA-related protein kinase CNK2, the mono-dehydroascorbate reductase MDAR1, the phosphoglycerate mutase PGM5 etc.. CONCLUSION: We propose that our 3'-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.

Novel shuttle markers for nuclear transformation of the green alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii today is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of the HSP70A-RBCS2 hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in both Chlamydomonas and Escherichia coli would also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of the Chlamydomonas genomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of a petC mutant with a modified BAC carrying the AphVIII marker along with the PETC gene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.

Structure and function of a novel endonuclease acting on branched DNA substrates.

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3' and 5' extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure-specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.

The structure of an archaeal homodimeric ligase which has RNA circularization activity.

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 A of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.

A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen.

During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.