Dmitrii E. Polev

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes.

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells.

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel‐enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure–function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose‐binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60–65 °C, respectively). Five crystal structures, with and without bound thio‐oligosaccharides, show conformational diversity of tunnel‐enclosing loops, including a form with partial tunnel collapse at subsite –4 not reported previously in GH7. Also, the first O‐glycosylation site in a GH7 crystal structure is reported – on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo‐initiation probability, similar to HjeCel7A.

Draft Genome Sequence of Geotrichum candidum Strain 3C

ABSTRACT We report here the draft genome sequence of Geotrichum candidum strain 3C, which is a filamentous yeast-like fungus that holds great promise for biotechnology. The genome was sequenced using Ion Torrent and 454 platforms. The estimated genome size was 41.4 Mb, and 14,579 protein-coding genes were predicted ab initio.

Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection

The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles.

Catching hidden variation: systematic correction of reference minor allele annotation in clinical variant calling

PurposeWe comprehensively assessed the influence of reference minor alleles (RMAs), one of the inherent problems of the human reference genome sequence.MethodsThe variant call format (VCF) files provided by the 1000 Genomes and Exome Aggregation Consortium (ExAC) consortia were used to identify RMA sites. All coding RMA sites were checked for concordance with UniProt and the presence of same codon variants. RMA-corrected predictions of functional effect were obtained with SIFT, PolyPhen-2, and PROVEAN standalone tools and compared with dbNSFP v2.9 for consistency.ResultsWe systematically characterized the problem of RMAs and identified several possible ways in which RMA could interfere with accurate variant discovery and annotation. We have discovered a systematic bias in the automated variant effect prediction at the RMA loci, as well as widespread switching of functional consequences for variants located in the same codon as the RMA. As a convenient way to address the problem of RMAs we have developed a simple bioinformatic tool that identifies variation at RMA sites and provides correct annotations for all such substitutions. The tool is available free of charge at http://rmahunter.bioinf.me.ConclusionCorrection of RMA annotation enhances the accuracy of next-generation sequencing–based methods in clinical practice.

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell’s history and to acquisition of new mutations near original break.

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage

Next generation DNA sequencing technologies are rapidly transforming the world of human genomics. Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are still frequently debated. In our study we developed a set of statistical tools to systematically assess coverage of CDS regions provided by several modern WES platforms, as well as PCR-free WGS. Using several novel metrics to characterize exon coverage in WES and WGS, we showed that some of the WES platforms achieve substantially less biased CDS coverage than others, with lower within- and between-interval variation and virtually absent GC-content bias. We discovered that, contrary to a common view, most of the coverage bias in WES stems from mappability limitations of short reads, as well as exome probe design. We identified the ~ 500 kb region of human exome that could not be effectively characterized using short read technology. We also showed that the overall power for SNP and indel discovery in CDS region is virtually indistinguishable for WGS and best WES platforms. Our results indicate that deep WES (100x) using least biased technologies provides similar effective coverage (97% of 10x q10+ bases) and CDS variant discovery to the standard 30x WGS, suggesting that WES remains an efficient alternative to WGS in many applications. Our work could serve as a guide for selection of an up-to-date resequencing approach in human genomic studies.

Transcriptome of the bivalve Limecola balthica L. from Western Pacific: A new resource for studies of European populations

The Baltic clam Limecola balthica L. (Tellinidae) is broadly used in ecophysiological, toxicological, evolutionary and environmental monitoring studies. However, it is poorly studied in respect of genome and gene functions. We obtained a transcriptome of Limecola b. balthica from Kamchatka (Western Pacific) generated with the use of Illumina high-throughput sequencing. We annotated 11,374 proteins, including 53 from the oxidative phosphorylation pathway and a number of pollution-stress biomarkers, recovered 254,540 single nucleotide variants within two annotated transcriptomes including 25,330 scorable in the previously published European data. Our results confirmed the available allozyme data indicating that nuclear genomes of the clams from the Baltic Sea were intermediate in their genetic composition between the Pacific (L. b. balthica) and the Atlantic (L. b. rubra) subspecies. At the same time, the mitochondrial genomes of Limecola from Kamchatka were nearly identical to the single published genome from the Baltic. The genomic diversity in Limecola was found to be high and comparable with that of other marine mollusks (0.0138 and 0.0142 heterozygous positions in the two studied transcriptomes). The data obtained in our study are a valuable resource for further development of genomic markers for evolutionary genetic and ecophysiological studies of L. balthica complex.

Mitochondrial Genome of Vannella croatica (Amoebozoa, Discosea, Vannellida)

Mitochondrial genome sequence of Vannella croatica (Amoebozoa, Discosea, Vannellida) was obtained using pulse‐field gel electrophoretic isolation of the circular mitochondrial DNA, followed by the next‐generation sequencing. The mitochondrial DNA of this species has the length of 28,933 bp and contains 12 protein‐coding genes, two ribosomal RNAs, and 16 transfer RNAs. Vannella croatica mitochondrial genome is relatively short compared to other known amoebozoan mitochondrial genomes but is rather gene‐rich and contains significant number of open reading frames.

Scytalidium candidum 3C is a new name for the Geotrichum candidum Link 3C strain

In the 1970s, the strain Geotrichum candidum Link 3C was isolated from rotting rope and since then has been extensively studied as a source of cellulose and xylan‐degrading enzymes. The original identification of the strain was based only on morphological characters of the fungal mycelium in culture. Recent comparison of the internal transcribed spacer (ITS) fragments derived from the draft genome published in 2015 did not show its similarity to G. candidum species. Given the value of the strain 3C in lignocellulosic biomass degradation, we performed morphological and molecular studies to find the appropriate taxonomic placement for this fungal strain within the Ascomycota phylum. ITS, 18S rDNA, 28S rDNA sequences, and RPB2 encoding genes were used to construct phylogenetic trees with Maximum likelihood and Bayesian inference methods. Based on sequence comparison and multiple gene sequencing, we conclude that the fungal strain designated as Geotrichum candidum Link 3C should be placed into the genus Scytalidium (Pezizomycotina, Leotiomycetes) and is redescribed herein as Scytalidium candidum 3C comb. nov.