Surgical Tumor Resection Deregulates Hallmarks of Cancer in Resected Tissue and the Surrounding Microenvironment.

Surgery exposes tumor tissue to severe hypoxia and mechanical stress leading to rapid gene expression changes in the tumor and its microenvironment, which remain poorly characterized. We biopsied tumor and adjacent normal tissues from patients with breast (n = 81) and head/neck squamous cancers (HNSC; n = 10) at the beginning (A), during (B), and end of surgery (C). Tumor/normal RNA from 46/81 patients with breast cancer was subjected to mRNA-Seq using Illumina short-read technology, and from nine patients with HNSC to whole-transcriptome microarray with Illumina BeadArray. Pathways and genes involved in 7 of 10 known cancer hallmarks, namely, tumor-promoting inflammation (TNF-A, NFK-B, IL18 pathways), activation of invasion and migration (various extracellular matrix-related pathways, cell migration), sustained proliferative signaling (K-Ras Signaling), evasion of growth suppressors (P53 signaling, regulation of cell death), deregulating cellular energetics (response to lipid, secreted factors, and adipogenesis), inducing angiogenesis (hypoxia signaling, myogenesis), and avoiding immune destruction (CTLA4 and PDL1) were significantly deregulated during surgical resection (time points A vs. B vs. C). These findings were validated using NanoString assays in independent pre/intra/post-operative breast cancer samples from 48 patients. In a comparison of gene expression data from biopsy (analogous to time point A) with surgical resection samples (analogous to time point C) from The Cancer Genome Atlas study, the top deregulated genes were the same as identified in our analysis, in five of the seven studied cancer types. This study suggests that surgical extirpation deregulates the hallmarks of cancer in primary tumors and adjacent normal tissue across different cancers. IMPLICATIONS: Surgery deregulates hallmarks of cancer in human tissue.

Building flexible and robust analysis frameworks for molecular subtyping of cancers.

Molecular subtyping is essential to infer tumor aggressiveness and predict prognosis. In practice, tumor profiling requires in-depth knowledge of bioinformatics tools involved in the processing and analysis of the generated data. Additionally, data incompatibility (e.g., microarray versus RNA sequencing data) and technical and uncharacterized biological variance between training and test data can pose challenges in classifying individual samples. In this article, we provide a roadmap for implementing bioinformatics frameworks for molecular profiling of human cancers in a clinical diagnostic setting. We describe a framework for integrating several methods for quality control, normalization, batch correction, classification and reporting, and develop a use case of the framework in breast cancer.

The mutational landscape of early- and typical-onset oral tongue squamous cell carcinoma.

BACKGROUND: The incidence of oral tongue squamous cell carcinoma (OTSCC) is increasing among younger birth cohorts. The etiology of early-onset OTSCC (diagnosed before the age of 50 years) and cancer driver genes remain largely unknown. METHODS: The Sequencing Consortium of Oral Tongue Cancer was established through the pooling of somatic mutation data of oral tongue cancer specimens (n = 227 [107 early-onset cases]) from 7 studies and The Cancer Genome Atlas. Somatic mutations at microsatellite loci and Catalog of Somatic Mutations in Cancer mutation signatures were identified. Cancer driver genes were identified with the MutSigCV and WITER algorithms. Mutation comparisons between early- and typical-onset OTSCC were evaluated via linear regression with adjustments for patient-related factors. RESULTS: Two novel driver genes (ATXN1 and CDC42EP1) and 5 previously reported driver genes (TP53, CDKN2A, CASP8, NOTCH1, and FAT1) were identified. Six recurrent mutations were identified, with 4 occurring in TP53. Early-onset OTSCC had significantly fewer nonsilent mutations even after adjustments for tobacco use. No associations of microsatellite locus mutations and mutation signatures with the age of OTSCC onset were observed. CONCLUSIONS: This international, multicenter consortium is the largest study to characterize the somatic mutational landscape of OTSCC and the first to suggest differences by age of onset. This study validates multiple previously identified OTSCC driver genes and proposes 2 novel cancer driver genes. In analyses by age, early-onset OTSCC had a significantly smaller somatic mutational burden that was not explained by differences in tobacco use. LAY SUMMARY: This study identifies 7 specific areas in the human genetic code that could be responsible for promoting the development of tongue cancer. Tongue cancer in young patients (under the age of 50 years) has fewer overall changes to the genetic code in comparison with tongue cancer in older patients, but the authors do not think that this is due to differences in smoking rates between the 2 groups. The cause of increasing cases of tongue cancer in young patients remains unclear.

Functional genomics screen with pooled shRNA library and gene expression profiling with extracts of Azadirachta indica identify potential pathways for therapeutic targets in head and neck squamous cell carcinoma.

Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-ß pathway and HSF-1-related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor.

Detection of High-Risk Human Papillomavirus in Oral Cavity Squamous Cell Carcinoma Using Multiple Analytes and Their Role in Patient Survival.

PURPOSE: Accurate detection of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) is essential to understanding the role of HPV in disease prognosis and management of patients. We used different analytes and methods to understand the true prevalence of HPV in a cohort of patients with OSCC with different molecular backgrounds, and we correlated HPV data with patient survival. METHODS: We integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, polymerase chain reaction [PCR], quantitative PCR [qPCR], and digital PCR), and molecular changes (somatic mutations and DNA methylation) from 153 patients with OSCC to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival. RESULTS: High prevalence (33% to 58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive and only 6% were both HPV DNA and HPV RNA positive. Most tumors with relatively high copy number HPV DNA and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA, and HPV RNA, either alone or in combination, did not correlate with patient survival. Nine HPV-associated genes stratified the virus-positive from the virus-negative tumor group with high confidence ( P < .008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity, and not HPV DNA alone, irrespective of copy number ( P < .2). CONCLUSION: In OSCC, the presence of both HPV RNA and p16 is rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore may not be informative. HPV DNA, HPV RNA, and p16 do not correlate with patients' outcome.

A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma.

Selection of the right reference gene(s) is crucial in the analysis and interpretation of gene expression data. The aim of the present study was to discover and validate a minimal set of internal control genes in head and neck tumor studies. We analyzed data from multiple sources (in house whole-genome gene expression microarrays, previously published quantitative real-time PCR (qPCR) data and RNA-seq data from TCGA) to come up with a list of 18 genes (discovery set) that had the lowest variance, a high level of expression across tumors, and their matched normal samples. The genes in the discovery set were ranked using four different algorithms (BestKeeper, geNorm, NormFinder, and comparative delta Ct) and a web-based comparative tool, RefFinder, for their stability and variance in expression across tissues. Finally, we validated their expression using qPCR in an additional set of tumor:matched normal samples that resulted in five genes (RPL30, RPL27, PSMC5, MTCH1, and OAZ1), out of which RPL30 and RPL27 were most stable and were abundantly expressed across the tissues. Our data suggest that RPL30 or RPL27 in combination with either PSMC5 or MTCH1 or OAZ1 can be used as a minimal set of control genes in head and neck tumor gene expression studies.

CAFE MOCHA: An Integrated Platform for Discovering Clinically Relevant Molecular Changes in Cancer-An Example of Distant Metastasis- and Recurrence-Linked Classifiers in Head and Neck Squamous Cell Carcinoma.

PURPOSE: With large amounts of multidimensional molecular data on cancers generated and deposited into public repositories such as The Cancer Genome Atlas and International Cancer Genome Consortium, a cancer type agnostic and integrative platform will help to identify signatures with clinical relevance. We devised such a platform and showcase it by identifying a molecular signature for patients with metastatic and recurrent (MR) head and neck squamous cell carcinoma (HNSCC). METHODS: We devised a statistical framework accompanied by a graphical user interface-driven application, Clinical Association of Functionally Established MOlecular CHAnges ( CAFE MOCHA; https://github.com/binaypanda/CAFEMOCHA), to discover molecular signatures linked to a specific clinical attribute in a cancer type. The platform integrates mutations and indels, gene expression, DNA methylation, and copy number variations to discover a classifier first and then to predict an incoming tumor for the same by pulling defined class variables into a single framework that incorporates a coordinate geometry-based algorithm called complete specificity margin-based clustering, which ensures maximum specificity. CAFE MOCHA classifies an incoming tumor sample using either its matched normal or a built-in database of normal tissues. The application is packed and deployed using the install4j multiplatform installer. We tested CAFE MOCHA in HNSCC tumors (n = 513) followed by validation in tumors from an independent cohort (n = 18) for discovering a signature linked to distant MR. RESULTS: CAFE MOCHA identified an integrated signature, MR44, associated with distant MR HNSCC, with 80% sensitivity and 100% specificity in the discovery stage and 100% sensitivity and 100% specificity in the validation stage. CONCLUSION: CAFE MOCHA is a cancer type and clinical attribute agnostic statistical framework to discover integrated molecular signatures.

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii.

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell's viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell's viper studied previously, we found 45-100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers.

A Minimal DNA Methylation Signature in Oral Tongue Squamous Cell Carcinoma Links Altered Methylation with Tumor Attributes.

UNLABELLED: Oral tongue squamous cell carcinomas (OTSCC) are a homogenous group of aggressive tumors in the head and neck region that spread early to lymph nodes and have a higher incidence of regional failure. In addition, there is a rising incidence of oral tongue cancer in younger populations. Studies on functional DNA methylation changes linked with altered gene expression are critical for understanding the mechanisms underlying tumor development and metastasis. Such studies also provide important insight into biomarkers linked with viral infection, tumor metastasis, and patient survival in OTSCC. Therefore, we performed genome-wide methylation analysis of tumors (N = 52) and correlated altered methylation with differential gene expression. The minimal tumor-specific DNA 5-methylcytosine signature identified genes near 16 different differentially methylated regions, which were validated using genomic data from The Cancer Genome Atlas cohort. In our cohort, hypermethylation of MIR10B was significantly associated with the differential expression of its target genes NR4A3 and BCL2L11 (P = 0.0125 and P = 0.014, respectively), which was inversely correlated with disease-free survival (P = 9E-15 and P = 2E-15, respectively) in patients. Finally, differential methylation in FUT3, TRIM5, TSPAN7, MAP3K8, RPS6KA2, SLC9A9, and NPAS3 genes was found to be predictive of certain clinical and epidemiologic parameters. IMPLICATIONS: This study reveals a functional minimal methylation profile in oral tongue tumors with associated risk habits, clinical, and epidemiologic outcomes. In addition, NR4A3 downregulation and correlation with patient survival suggests a potential target for therapeutic intervention in oral tongue tumors. Data from the current study are deposited in the NCBI Geo database (accession number GSE75540). Mol Cancer Res; 14(9); 805-19. ©2016 AACR.

An Improved Genome Assembly of Azadirachta indica A. Juss.

Neem (Azadirachta indica A. Juss.), an evergreen tree of the Meliaceae family, is known for its medicinal, cosmetic, pesticidal and insecticidal properties. We had previously sequenced and published the draft genome of a neem plant, using mainly short read sequencing data. In this report, we present an improved genome assembly generated using additional short reads from Illumina and long reads from Pacific Biosciences SMRT sequencer. We assembled short reads and error-corrected long reads using Platanus, an assembler designed to perform well for heterozygous genomes. The updated genome assembly (v2.0) yielded 3- and 3.5-fold increase in N50 and N75, respectively; 2.6-fold decrease in the total number of scaffolds; 1.25-fold increase in the number of valid transcriptome alignments; 13.4-fold less misassembly and 1.85-fold increase in the percentage repeat, over the earlier assembly (v1.0). The current assembly also maps better to the genes known to be involved in the terpenoid biosynthesis pathway. Together, the data represent an improved assembly of the A. indica genome.

Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits, HPV, clinical parameters and tumor recurrence.

Oral tongue squamous cell carcinomas (OTSCC) are a homogeneous group of tumors characterized by aggressive behavior, early spread to lymph nodes and a higher rate of regional failure. Additionally, the incidence of OTSCC among younger population (<50yrs) is on the rise; many of whom lack the typical associated risk factors of alcohol and/or tobacco exposure. We present data on single nucleotide variations (SNVs), indels, regions with loss of heterozygosity (LOH), and copy number variations (CNVs) from fifty-paired oral tongue primary tumors and link the significant somatic variants with clinical parameters, epidemiological factors including human papilloma virus (HPV) infection and tumor recurrence. Apart from the frequent somatic variants harbored in TP53, CASP8, RASA1, NOTCH and CDKN2A genes, significant amplifications and/or deletions were detected in chromosomes 6-9, and 11 in the tumors. Variants in CASP8 and CDKN2A were mutually exclusive. CDKN2A, PIK3CA, RASA1 and DMD variants were exclusively linked to smoking, chewing, HPV infection and tumor stage. We also performed a whole-genome gene expression study that identified matrix metalloproteases to be highly expressed in tumors and linked pathways involving arachidonic acid and NF-k-B to habits and distant metastasis, respectively. Functional knockdown studies in cell lines demonstrated the role of CASP8 in a HPV-negative OTSCC cell line. Finally, we identified a 38-gene minimal signature that predicts tumor recurrence using an ensemble machine-learning method. Taken together, this study links molecular signatures to various clinical and epidemiological factors in a homogeneous tumor population with a relatively high HPV prevalence.

Augmenting transcriptome assembly by combining de novo and genome-guided tools.

Researchers interested in studying and constructing transcriptomes, especially for non-model species, face the conundrum of choosing from a number of available de novo and genome-guided assemblers. None of the popular assembly tools in use today achieve requisite sensitivity, specificity or recovery of full-length transcripts on their own. Here, we present a comprehensive comparative study of the performance of various assemblers. Additionally, we present an approach to combinatorially augment transciptome assembly by using both de novo and genome-guided tools. In our study, we obtained the best recovery and most full-length transcripts with Trinity and TopHat1-Cufflinks, respectively. The sensitivity of the assembly and isoform recovery was superior, without compromising much on the specificity, when transcripts from Trinity were augmented with those from TopHat1-Cufflinks.

COPS: a sensitive and accurate tool for detecting somatic Copy Number Alterations using short-read sequence data from paired samples.

Copy Number Alterations (CNAs) such as deletions and duplications; compose a larger percentage of genetic variations than single nucleotide polymorphisms or other structural variations in cancer genomes that undergo major chromosomal re-arrangements. It is, therefore, imperative to identify cancer-specific somatic copy number alterations (SCNAs), with respect to matched normal tissue, in order to understand their association with the disease. We have devised an accurate, sensitive, and easy-to-use tool, COPS, COpy number using Paired Samples, for detecting SCNAs. We rigorously tested the performance of COPS using short sequence simulated reads at various sizes and coverage of SCNAs, read depths, read lengths and also with real tumor:normal paired samples. We found COPS to perform better in comparison to other known SCNA detection tools for all evaluated parameters, namely, sensitivity (detection of true positives), specificity (detection of false positives) and size accuracy. COPS performed well for sequencing reads of all lengths when used with most upstream read alignment tools. Additionally, by incorporating a downstream boundary segmentation detection tool, the accuracy of SCNA boundaries was further improved. Here, we report an accurate, sensitive and easy to use tool in detecting cancer-specific SCNAs using short-read sequence data. In addition to cancer, COPS can be used for any disease as long as sequence reads from both disease and normal samples from the same individual are available. An added boundary segmentation detection module makes COPS detected SCNA boundaries more specific for the samples studied. COPS is available at ftp://115.119.160.213 with username "cops" and password "cops".

A draft of the genome and four transcriptomes of a medicinal and pesticidal angiosperm Azadirachta indica.

BACKGROUND: The Azadirachta indica (neem) tree is a source of a wide number of natural products, including the potent biopesticide azadirachtin. In spite of its widespread applications in agriculture and medicine, the molecular aspects of the biosynthesis of neem terpenoids remain largely unexplored. The current report describes the draft genome and four transcriptomes of A. indica and attempts to contextualise the sequence information in terms of its molecular phylogeny, transcript expression and terpenoid biosynthesis pathways. A. indica is the first member of the family Meliaceae to be sequenced using next generation sequencing approach. RESULTS: The genome and transcriptomes of A. indica were sequenced using multiple sequencing platforms and libraries. The A. indica genome is AT-rich, bears few repetitive DNA elements and comprises about 20,000 genes. The molecular phylogenetic analyses grouped A. indica together with Citrus sinensis from the Rutaceae family validating its conventional taxonomic classification. Comparative transcript expression analysis showed either exclusive or enhanced expression of known genes involved in neem terpenoid biosynthesis pathways compared to other sequenced angiosperms. Genome and transcriptome analyses in A. indica led to the identification of repeat elements, nucleotide composition and expression profiles of genes in various organs. CONCLUSIONS: This study on A. indica genome and transcriptomes will provide a model for characterization of metabolic pathways involved in synthesis of bioactive compounds, comparative evolutionary studies among various Meliaceae family members and help annotate their genomes. A better understanding of molecular pathways involved in the azadirachtin synthesis in A. indica will pave ways for bulk production of environment friendly biopesticides.

Chimeras of Escherichia coli and Mycobacterium tuberculosis single-stranded DNA binding proteins: characterization and function in Escherichia coli.

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mß1, mß1'ß2, mß1-ß5, mß1-ß6 and mß4-ß5) by transplanting ß1, ß1'ß2, ß1-ß5, ß1-ß6 and ß4-ß5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mß1'ß2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the ß2 strand of mß1'ß2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mß1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mß1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mß1-ß6, MtuSSB, mß1'ß2 and mß1-ß5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mß1-ß5 SSB was poor. In contrast, mß1'ß2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

A comparative approach to elucidate chloroplast genome replication.

BACKGROUND: Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts. RESULTS: We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication. CONCLUSION: Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population.

Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation.

BACKGROUND: Human Rad51 (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA) in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPgammaS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding. RESULTS: We find that human Rad52 (RAD52) stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, (except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction) while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding. CONCLUSION: RAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

BACKGROUND: Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. RESULTS: We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. CONCLUSION: Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.

Mitochondrial tRNA sequences as unusual replication origins: pathogenic implications for Homo sapiens.

The heavy strand of vertebrate mitochondrial genomes accumulates deaminations proportionally to the time it spends single-stranded during replication. A previous study showed that the strength of genome-wide deamination gradients originating from tRNA gene's locations increases with their capacities to form secondary structures resembling mitochondrial origins of light strand replication (OL), suggesting an alternative function for tRNA sequences. We hypothesize that this function is frequently pathogenic for those tRNA genes that normally do not form OL-like structures, because this could cause excess mutations in genome regions unadapted to tolerate them. In human mitochondrial genomes, pathogenic tRNA variants usually form less OL-like structures than non-pathogenic ones in cases where the normal non-pathogenic tRNA variant can function as OL, as evolutionary analyses reveal. For tRNAs lacking the putative OL-like functioning capacity, pathogenic variants form more OL-like secondary structures, particularly structures that might invoke bi-directional replication (true for 14 among 21 tRNA species, p<0.05, sign test; significantly at p<0.05 (1 tailed test) for 7 tRNA species), but not more unidirectional replication invoking structures. Accounting for the functional cloverleaf-like structure-forming capacities of tRNAs yields similar results. Rare, non-pathogenic tRNA mutants tend to form more OL-like structures than the common, non-pathogenic ones, suggesting weak directional selection also among non-pathogenic variants. The duration spent single stranded by a region of the heavy strand (D(ssH)) during replication, estimated by integrating over all regions that can function as OL in Homo sapiens mitochondrial genomes, increases with distance of that region from the Dloop. This suggests convergence of single-strandedness during replication and transcription, and explains conserved locations of tRNA species in mitochondrial genomes and bacterial operons. These locations minimize deamination costs only in anticodons and not in other tRNA regions, during replication and transcription. Therefore, putative functioning as OLs by tRNA sequences is normal at some locations and pathogenic at others.

Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in lizards.

Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.