Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

PHACTboost: A Phylogeny-aware Pathogenicity Predictor for the Missense Mutations via Boosting.

Most algorithms that are used to predict the effects of variants rely on evolutionary conservation. However, a majority of such techniques compute evolutionary conservation by solely using the alignment of multiple sequences while overlooking the evolutionary context of substitution events. We had introduced PHACT, a scoring-based pathogenicity predictor for missense mutations that can leverage phylogenetic trees, in our previous study. By building on this foundation, we now propose PHACTboost, a gradient boosting tree-based classifier that combines PHACT scores with information from multiple sequence alignments, phylogenetic trees, and ancestral reconstruction. By learning from data PHACTboost outperforms PHACT. Furthermore, the results of comprehensive experiments on carefully constructed sets of variants demonstrated that PHACTboost can outperform 40 prevalent pathogenicity predictors reported in the dbNSFP, including conventional tools, meta-predictors, and deep learning-based approaches as well as more recent tools such as, AlphaMissense, EVE, and CPT-1. The superiority of PHACTboost over these methods was particularly evident in case of hard variants for which different pathogenicity predictors offered conflicting results. We provide predictions of 215 million amino acid alterations over 20,191 proteins. PHACTboost is available at https://github.com/CompGenomeLab/PHACTboost. PHACTboost can improve our understanding of genetic diseases and facilitate more accurate diagnoses.

Effects of replication domains on genome-wide UV-induced DNA damage and repair.

Nucleotide excision repair is the primary repair mechanism that removes UV-induced DNA lesions in placentals. Unrepaired UV-induced lesions could result in mutations during DNA replication. Although the mutagenesis of pyrimidine dimers is reasonably well understood, the direct effects of replication fork progression on nucleotide excision repair are yet to be clarified. Here, we applied Damage-seq and XR-seq techniques and generated replication maps in synchronized UV-treated HeLa cells. The results suggest that ongoing replication stimulates local repair in both early and late replication domains. Additionally, it was revealed that lesions on lagging strand templates are repaired slower in late replication domains, which is probably due to the imbalanced sequence context. Asymmetric relative repair is in line with the strand bias of melanoma mutations, suggesting a role of exogenous damage, repair, and replication in mutational strand asymmetry.

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila.

Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

The interplay of 3D genome organization with UV-induced DNA damage and repair.

The 3D organization of the eukaryotic genome is crucial for various cellular processes such as gene expression and epigenetic regulation, as well as for maintaining genome integrity. However, the interplay between UV-induced DNA damage and repair with the 3D structure of the genome is not well understood. Here, we used state-of-the-art Hi-C, Damage-seq, and XR-seq datasets and in silico simulations to investigate the synergistic effects of UV damage and 3D genome organization. Our findings demonstrate that the peripheral 3D organization of the genome shields the central regions of genomic DNA from UV-induced damage. Additionally, we observed that potential damage sites of pyrimidine-pyrimidone (6-4) photoproducts are more prevalent in the nucleus center, possibly indicating an evolutionary pressure against those sites at the periphery. Interestingly, we found no correlation between repair efficiency and 3D structure after 12 min of irradiation, suggesting that UV radiation alters the genome's 3D organization in a short period of time. Interestingly, however, 2 h after UV induction, we observed more efficient repair levels in the center of the nucleus relative to the periphery. These results have implications for understanding the etiology of cancer and other diseases, as the interplay between UV radiation and the 3D genome may play a role in the development of genetic mutations and genomic instability.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Discovering misannotated lncRNAs using deep learning training dynamics.

MOTIVATION: Recent experimental evidence has shown that some long non-coding RNAs (lncRNAs) contain small open reading frames (sORFs) that are translated into functional micropeptides, suggesting that these lncRNAs are misannotated as non-coding. Current methods to detect misannotated lncRNAs rely on ribosome-profiling (Ribo-Seq) and mass-spectrometry experiments, which are cell-type dependent and expensive. RESULTS: Here, we propose a computational method to identify possible misannotated lncRNAs from sequence information alone. Our approach first builds deep learning models to discriminate coding and non-coding transcripts and leverages these models' training dynamics to identify misannotated lncRNAs-i.e. lncRNAs with coding potential. The set of misannotated lncRNAs we identified significantly overlap with experimentally validated ones and closely resemble coding protein sequences as evidenced by significant BLAST hits. Our analysis on a subset of misannotated lncRNA candidates also shows that some ORFs they contain yield high confidence folded structures as predicted by AlphaFold2. This methodology offers promising potential for assisting experimental efforts in characterizing the hidden proteome encoded by misannotated lncRNAs and for curating better datasets for building coding potential predictors. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/nabiafshan/DetectingMisannotatedLncRNAs. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PHACT: Phylogeny-Aware Computing of Tolerance for Missense Mutations.

Evolutionary conservation is a fundamental resource for predicting the substitutability of amino acids and the loss of function in proteins. The use of multiple sequence alignment alone-without considering the evolutionary relationships among sequences-results in the redundant counting of evolutionarily related alteration events, as if they were independent. Here, we propose a new method, PHACT, that predicts the pathogenicity of missense mutations directly from the phylogenetic tree of proteins. PHACT travels through the nodes of the phylogenetic tree and evaluates the deleteriousness of a substitution based on the probability differences of ancestral amino acids between neighboring nodes in the tree. Moreover, PHACT assigns weights to each node in the tree based on their distance to the query organism. For each potential amino acid substitution, the algorithm generates a score that is used to calculate the effect of substitution on protein function. To analyze the predictive performance of PHACT, we performed various experiments over the subsets of two datasets that include 3,023 proteins and 61,662 variants in total. The experiments demonstrated that our method outperformed the widely used pathogenicity prediction tools (i.e., SIFT and PolyPhen-2) and achieved a better predictive performance than other conventional statistical approaches presented in dbNSFP. The PHACT source code is available at https://github.com/CompGenomeLab/PHACT.

MiST 3.0: an updated microbial signal transduction database with an emphasis on chemosensory systems.

Bacteria and archaea employ dedicated signal transduction systems that modulate gene expression, second-messenger turnover, quorum sensing, biofilm formation, motility, host-pathogen and beneficial interactions. The updated MiST database provides a comprehensive classification of microbial signal transduction systems. This update is a result of a substantial scaling to accommodate constantly growing microbial genomic data. More than 125 000 genomes, 516 million genes and almost 100 million unique protein sequences are currently stored in the database. For each bacterial and archaeal genome, MiST 3.0 provides a complete signal transduction profile, thus facilitating theoretical and experimental studies on signal transduction and gene regulation. New software infrastructure and distributed pipeline implemented in MiST 3.0 enable regular genome updates based on the NCBI RefSeq database. A novel MiST feature is the integration of unique profile HMMs to link complex chemosensory systems with corresponding chemoreceptors in bacterial and archaeal genomes. The data can be explored online or via RESTful API (freely available at https://mistdb.com).

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events.

BACKGROUND: Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. RESULTS: We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. CONCLUSION: Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Single-nucleotide resolution dynamic repair maps of UV damage in Saccharomyces cerevisiae genome.

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5' and 6-7 nucleotides 3' to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.

Cisplatin-DNA adduct repair of transcribed genes is controlled by two circadian programs in mouse tissues.

Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene's phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.

Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells.

Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.

Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase.

We used high-throughput sequencing of short, cyclobutane pyrimidine dimer-containing ssDNA oligos generated during repair of UV-induced damage to study that process at both mechanistic and systemic levels in Escherichia coli Numerous important insights on DNA repair were obtained, bringing clarity to the respective roles of UvrD helicase and Mfd translocase in repair of UV-induced damage. Mechanistically, experiments showed that the predominant role of UvrD in vivo is to unwind the excised 13-mer from dsDNA and that mutation of uvrD results in remarkable protection of that oligo from exonuclease activity as it remains hybridized to the dsDNA. Genome-wide analysis of the transcribed strand/nontranscribed strand (TS/NTS) repair ratio demonstrated that deletion of mfd globally shifts the distribution of TS/NTS ratios downward by a factor of about 2 on average for the most highly transcribed genes. Even for the least transcribed genes, Mfd played a role in preferential repair of the transcribed strand. On the other hand, mutation of uvrD, if anything, slightly pushed the distribution of TS/NTS ratios to higher ratios. These results indicate that Mfd is the transcription repair-coupling factor whereas UvrD plays a role in excision repair by aiding the catalytic turnover of excision repair proteins.

Dynamic maps of UV damage formation and repair for the human genome.

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. BaP forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors, and after damage-specific immunoprecipitation, the adaptor-ligated oligonucleotides are converted to dsDNA with an appropriate translesion DNA synthesis (TLS) polymerase, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method translesion excision repair-sequencing (tXR-seq). In contrast to our previously described XR-seq method, tXR-seq does not depend on repair/removal of the damage in the excised oligonucleotides, and thus it is applicable to essentially all DNA damages processed by nucleotide excision repair. Here we present the excision repair maps for CPDs and BPDE-dG adducts generated by tXR-Seq for the human genome. In addition, we report the sequence specificity of BPDE-dG excision repair using tXR-seq.

Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli.

Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA-spoT- mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli.

Molecular mechanisms and genomic maps of DNA excision repair in Escherichia coli and humans.

Nucleotide excision repair is a major DNA repair mechanism in all cellular organisms. In this repair system, the DNA damage is removed by concerted dual incisions bracketing the damage and at a precise distance from the damage. Here, we review the basic mechanisms of excision repair in Escherichia coli and humans and the recent genome-wide mapping of DNA damage and repair in these organisms at single-nucleotide resolution.

Class III Histidine Kinases: a Recently Accessorized Kinase Domain in Putative Modulators of Type IV Pilus-Based Motility.

Histidine kinases are key components of regulatory systems that enable bacteria to respond to environmental changes. Two major classes of histidine kinases are recognized on the basis of their modular design: classical (HKI) and chemotaxis specific (HKII). Recently, a new type of histidine kinase that appeared to have features of both HKIs and HKIIs was identified and termed HKIII; however, the details of HKIII's relationship to other two classes of histidine kinases, their function, and evolutionary history remain unknown. Here, we carried out genomic, phylogenetic, and protein sequence analyses that allowed us to reveal the unusual evolutionary history of this protein family, formalize its distinctive features, and propose its putative function. HKIIIs are characterized by the presence of sensory domains and the lack of a dimerization domain, which is typically present in all histidine kinases. In addition to a single-domain response regulator, HKIII signal transduction systems utilize CheX phosphatase and, in many instances, an unorthodox soluble chemoreceptor that are usual components of chemotaxis signal transduction systems. However, many HKIII genes are found in genomes completely lacking chemotaxis genes, thus decoupling their function from chemotaxis. By contrast, all HKIII-containing genomes also contain pilT, a marker gene for bacterial type IV pilus-based motility, whose regulation is proposed as a putative function for HKIII. These signal transduction systems have a narrow phyletic distribution but are present in many emerging and opportunistic pathogens, thus offering an attractive potential target for future antimicrobial drug design.IMPORTANCE Bacteria adapt to their environment and their hosts by detecting signals and regulating their cellular functions accordingly. Here, we describe a largely unexplored family of signal transduction histidine kinases, called HKIII, that have a unique modular design. While they are currently identified in a relatively short list of bacterial species, this list contains many emerging pathogens. We show that HKIIIs likely control bacterial motility across solid surfaces, which is a key virulence factor in many bacteria, including those causing severe infections. Full understanding of this putative function may help in designing effective drugs against pathogens that will not affect the majority of the beneficial human microbiome.

Aquerium: A web application for comparative exploration of domain-based protein occurrences on the taxonomically clustered genome tree.

Gene duplication and loss are major driving forces in evolution. While many important genomic resources provide information on gene presence, there is a lack of tools giving equal importance to presence and absence information as well as web platforms enabling easy visual comparison of multiple domain-based protein occurrences at once. Here, we present Aquerium, a platform for visualizing genomic presence and absence of biomolecules with a focus on protein domain architectures. The web server offers advanced domain organization querying against the database of pre-computed domains for ∼26,000 organisms and it can be utilized for identification of evolutionary events, such as fusion, disassociation, duplication, and shuffling of protein domains. The tool also allows alternative inputs of custom entries or BLASTP results for visualization. Aquerium will be a useful tool for biologists who perform comparative genomic and evolutionary analyses. The web server is freely accessible at http://aquerium.utk.edu. Proteins 2016; 85:72-77. © 2016 Wiley Periodicals, Inc.