DNA Secondary Structures Research Articles

A Comprehensive Genetic Analysis of Mycotoxin-Producing Penicillium expansum Isolated from River Water Using Molecular Profiling, DNA Barcoding, and Secondary Structure Prediction

This study marks the first report on the genetic characterization of Penicillium expansum strain capable of mycotoxin production isolated from river water. Situated in Ganagalawanipeta village, Srikakulam, Andhra Pradesh, India, where river water serves as a vital resource, our investigation probed the presence of pathogenic opportunistic fungi adept at mycotoxin synthesis. Over six months, 30 samples were collected to assess their occurrence. This article revolves around the use of morphological traits for Penicillium genus identification. Precise species determination involved PCR analysis using universal primers ITS1 and ITS4, followed by sequence analysis through NCBI-BLASTn and the ITS2 database. The analysis indicated a striking 99.49% genetic similarity to Penicillium expansum isolate MW559596 from CSIR-National Institute of Oceanography, Goa, an Indian isolate, with a resultant 600-base pair fragment. This sequence was officially cataloged as OR536221 in the NCBI GenBank database. Sequence and phylogenetic assessments were conducted to pinpoint the strain and geographical origin. Notably, the ribosomal nuclear ITS region displayed significant inter- and intra-specific divergence, manifested in DNA barcodes and secondary structures established via minimum free energy calculations. These findings provide crucial insights into the genetic diversity and potential mycotoxin production of P. expansum isolates, shedding light on the environmental repercussions and health risks associated with river water contamination from agricultural and aquaculture effluents. This pioneering research advances our understanding of mycotoxin-producing fungi in aquatic environments and underscores the imperative need for water quality monitoring in regions reliant on such water sources for their sustenance and livelihoods.

G-quadruplex stabilizer CX-5461 effectively combines with radiotherapy to target ATRX-deficient malignant glioma.

Inactivation of α-thalassaemia/mental retardation X-linked (ATRX) represents a defining molecular feature in large subsets of malignant glioma. ATRX deficiency gives rise to abnormal G-quadruplex (G4) DNA secondary structures, enhancing replication stress and genomic instability. Building on earlier work, we evaluated the extent to which pharmacological G4 stabilization selectively enhances DNA damage and cell death in ATRX-deficient preclinical glioma models. Using the G4 stabilizer CX-5461, we treated patient-derived glioma stem cells (GSCs) in vitro and GSC flank and intracranial murine xenografts in vivo to evaluate efficacy as both a single agent and in combination with ionizing radiation (IR), the latter a central element of current treatment standards. CX-5461 promoted dose-sensitive lethality in ATRX-deficient GSCs relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage, activated p53-independent apoptosis, and induced G2/M arrest to a greater extent in ATRX-deficient GSCs than in ATRX-intact counterparts. These data were corroborated in vivo, where CX-5461/IR treatment profoundly delayed tumor growth and prolonged survival in mice bearing ATRX-deficient flank xenografts. Histopathological analyses revealed decreased proliferation, increased apoptosis, and significant G4 induction, replication stress, and DNA damage in CX-5461-treated tumors, both alone and in combination with IR. Finally, despite suboptimal blood-brain-barrier penetration, systemic CX-5461 treatment induced tangible pharmacodynamic effects in ATRX-deficient intracranial GSC models. In totality, our work substantively demonstrates efficacy and defines mechanisms of action for G4 stabilization as a novel therapeutic strategy targeting ATRX-deficient malignant glioma, laying the groundwork for clinical translation.

Abstract PR020: A novel framework for epigenome-dependent multimodal fragment-signature analysis reveals insights into cell-free DNA generation in cancer

Abstract Background: The genome-wide pattern of cell-free DNA (cfDNA) is influenced by fragmentation enzymes, nucleosome occupancy, and epigenetic factors, as well as cell death mechanisms. Better modeling of these factors and their interactions will be essential for more sensitive detection of tumor-specific features and tissue-of-origin. Methods: To obtain mechanistic insights into cfDNA fragmentation, we developed a fragmentomics signature analysis framework. Our fragmenTopics algorithm (i) decomposes contributions of different fragment lengths and end-motif signatures using topic modeling and (ii) learns the dependencies of signature rates on the epigenome. The second component utilizes gradient boosting, enabling the modeling of non-linear dependencies and feature interactions. Both large-scale features, such as histone marks and DNase activity, and meso-scale features, such as transcription start sites (TSSs), nucleosome positions, and secondary DNA structures, are included in the model. Results: Using de novo signature discovery from 726 samples across five datasets, we built a comprehensive catalog of fragment length and end-motif signatures and studied determinants of their activity across the genome. The most common length and end-motif signatures reflected the mononucleosomal fragmentation by DNASE1L3. These signatures, as well as the dinucleotide length signature and motif signatures associated with known motifs of DNase enzymes, such as DFFB, showed lower abundance in cancer and further decreased with increasing tumor fraction. Instead, cancer samples manifested a higher diversity of processes, many of which are of unknown origin. Notably, two cancer-enriched length signatures were, on average, 20 bp shorter fragments with respect to mono- and di-nucleosome peaks. These signatures correlated with H3K4me1 and the compartment eigenvector, and may be linked to tighter nucleosome wrapping in euchromatin. Two cancer-enriched signatures (190-240 bp and 240-300 bp) had higher rates in TSSs; in addition to the two tight mono- and di-nucleosomal signatures, which were also enriched. Cancer-enriched end-motifs were A/T-rich, and these did not correspond to known DNase motifs. The increased diversity in end-motifs may be due to non-apoptotic cell death or tissue- or cancer-specific activity of less-studied cleavage enzymes; our results on their epigenome correlations can inform further investigations into their etiology. Conclusion: Our signature-based fragmentomics framework, which decouples the contributions of distinct processes while identifying the epigenomic determinants of their genome-wide rates, can improve our understanding of cell-free DNA fragmentation in cancer. Citation Format: Yoo-Na Kim, Sangmi Lee, Allen Lynch, Tae Hee Kim, Peter J Park, Doga Gulhan. A novel framework for epigenome-dependent multimodal fragment-signature analysis reveals insights into cell-free DNA generation in cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR020.

Comparative analysis of predicted DNA secondary structures infers complex human centromere topology

Secondary structures are non-canonical arrangements of nucleic acids due to intra-strand interactions, including base pairing, stacking, or other higher-order features that deviate from the standard double-helical conformation. While these structures are extensively studied in RNA, they can also form when DNA becomes single stranded, creating topological roadblocks that can impact essential DNA-based processes such as replication, transcription, and repair, ultimately affecting genome stability. The availability of a complete linear sequence of human genomes, including repetitive loci, enables the prediction of DNA secondary structures comparing across various regions. Here, we evaluate the intrinsic properties of linear single-stranded DNA sequences derived from sampling specialized human loci such as centromeres, pericentromeres, ribosomal DNA (rDNA), and coding regions from the CHM13 genome. Our comparative analysis of predicted secondary structures across human chromosomes revealed the heightened presence, complexity, and instability of secondary structures within the centromere, which gradually decreased toward the pericentromere onto chromosomes' arms, on average lowest in coding regions. Notably, centromeric repeats exhibited the highest level of topological complexity within both the active and divergent domains, even when compared to other repetitive tandem satellites, such as rDNA in acrocentric chromosomes. Our findings provide evidence of the intrinsic self-hybridizing properties of centromere repeats, which are capable of generating complex topological structures that may functionally correlate with chromosome missegregation, especially when centromeric chromatin is disrupted. Processes such as long non-coding RNA transcription, recombination, and other mechanisms that dechromatinize and unwind stretches of linear DNA in these regions create invivo opportunities for the DNA acrobatics hereby predicted.

G-Quadruplex Recognition by Tetraalkylammonium Ions: A New Paradigm for Discrimination between Parallel and Antiparallel G-Quadruplexes.

G-quadruplexes are an important class of noncanonical secondary structures of DNA that exist in the cell and are involved in the regulation of principal genomic events. Any regulatory role of a G-quadruplex in the genome is coupled with the attendant interconversions between the G-quadruplex and duplex states. Much effort has been invested in the quest for agents that can recognize individual G-quadruplexes and shift the associated duplex-G-quadruplex equilibria toward the G-quadruplex state. In this communication, we demonstrate that, notwithstanding their simplicity, tetraalkylammonium ions, [H(CH2)n]4N+, recognize and strongly stabilize the parallel c-MYC G-quadruplex, while not binding to the antiparallel human telomeric G-quadruplex or duplex DNA. The affinity of TAA+ ions for the c-MYC G-quadruplex correlates with the length of the aliphatic side chains. Our CD spectral data suggest that the binding of tetraalkylammonium ions is external, not resulting in structural changes in the host G-quadruplex. The observed discriminatory power identifies tetraalkylammonium salts as a starting point for developing topology-selective G-quadruplex-binding agents.

Optimization of 18 S rRNA metabarcoding for the simultaneous diagnosis of intestinal parasites

Recent advancements in next-generation sequencing (NGS) technologies have created new opportunities for comprehensive screening of multiple parasite species. In this study, we cloned the 18 S rDNA V9 region of 11 species of intestinal parasites into plasmids. Equal amounts and concentrations of these 11 plasmids were pooled, and amplicon NGS targeting the 18 S rDNA V9 region was performed using the Illumina iSeq 100 platform. A total of 434,849 reads were identified, and all 11 parasite species were detected, although the number of output reads for each parasite varied. The read count ratio, in descending order, was as follows: Clonorchis sinensis, 17.2%; Entamoeba histolytica, 16.7%; Dibothriocephalus latus, 14.4%; Trichuris trichiura, 10.8%; Fasciola hepatica, 8.7%; Necator americanus, 8.5%; Paragonimus westermani, 8.5%; Taenia saginata, 7.1%; Giardia intestinalis, 5.0%; Ascaris lumbricoides, 1.7%; and Enterobius vermicularis, 0.9%. We found that the DNA secondary structures showed a negative association with the number of output reads. Additionally, variations in the amplicon PCR annealing temperature affected the relative abundance of output reads for each parasite. These findings can be applied to improve parasite detection methodologies and ultimately enhance efforts to control and prevent intestinal parasitic infections.

Applying Machine Learning and SERS for Precise Typing of DNA Secondary Structures.

Surface-enhanced Raman spectroscopy (SERS) has been demonstrated as an effective method for elucidating secondary structural characteristics of DNA. However, the inherent complexity of the DNA conformation and the lack of SERS samples pose challenges for identifying numerous secondary structures. To address these issues, a synergistic method integrating machine learning with SERS was proposed so as to analyze the SERS spectra of 54 well-defined conformational oligonucleotides, namely, G-quadruplex (G4), i-motif (iM), double-strand (DS), and single-strand (SS) configurations. Principal component analysis (PCA) effectively segregated the oligonucleotides into three distinct conformational groups (G4s, iMs, and others). Furthermore, linear discriminant analysis (LDA), K-nearest neighbor (KNN), and support vector machine (SVM) approaches were utilized to improve the typing accuracy of 54 trained sequences. This enabled the correct classification of the structures of five untrained sequences, as well as the identification of the predominant conformations including G4, iM, and DS formed by two complementary G-rich and C-rich sequences in acidic and neutral pH conditions. The results of this study demonstrated the potential of the proposed methodology for rapid screening and prediction of secondary DNA conformations.

Mimicking a Cellular Crowding Environment for Enzyme-Free Paper-Based Nucleic Acid Tests at the Point of Care.

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases in the same location where the patient receives treatment. Communicable diseases, such as human immunodeficiency virus (HIV), disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource-intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes, producing large DNA nanostructures, capable of single-molecule detection in cellulo. These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here, we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub-pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an in cellulo reaction in vitro by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

Predicting bacterial transcription factor binding sites through machine learning and structural characterization based on DNA duplex stability.

Transcriptional factors (TFs) in bacteria play a crucial role in gene regulation by binding to specific DNA sequences, thereby assisting in the activation or repression of genes. Despite their central role, deciphering shape recognition of bacterial TFs-DNA interactions remains an intricate challenge. A deeper understanding of DNA secondary structures could greatly enhance our knowledge of how TFs recognize and interact with DNA, thereby elucidating their biological function. In this study, we employed machine learning algorithms to predict transcription factor binding sites (TFBS) and classify them as directed-repeat (DR) or inverted-repeat (IR). To accomplish this, we divided the set of TFBS nucleotide sequences by size, ranging from 8 to 20 base pairs, and converted them into thermodynamic data known as DNA duplex stability (DDS). Our results demonstrate that the Random Forest algorithm accurately predicts TFBS with an average accuracy of over 82% and effectively distinguishes between IR and DR with an accuracy of 89%. Interestingly, upon converting the base pairs of several TFBS-IR into DDS values, we observed a symmetric profile typical of the palindromic structure associated with these architectures. This study presents a novel TFBS prediction model based on a DDS characteristic that may indicate how respective proteins interact with base pairs, thus providing insights into molecular mechanisms underlying bacterial TFs-DNA interaction.

The first embryonic landscape of G-quadruplexes related to myogenesis

BackgroundDNA G-quadruplexes (G4s) represent a distinctive class of non-canonical DNA secondary structures. Despite their recognition as potential therapeutic targets in some cancers, the developmental role of G4 structures remains enigmatic. Mammalian embryonic myogenesis studies are hindered by limitations, prompting the use of chicken embryo-derived myoblasts as a model to explore G4 dynamics. This study aims to reveal the embryonic G4s landscape and elucidate the underlying mechanisms for candidate G4s that influence embryonic myogenesis.ResultsThis investigation unveils a significant reduction in G4s abundance during myogenesis. G4s stabilizer pyridostatin impedes embryonic myogenesis, emphasizing the regulatory role of G4s in this process. G4 Cut&Tag sequencing and RNA-seq analyses identify potential G4s and DEGs influencing embryonic myogenesis. Integration of G4 and DEG candidates identifies 32 G4s located in promoter regions capable of modulating gene transcription. WGBS elucidates DNA methylation dynamics during embryonic myogenesis. Coordinating transcriptome data with DNA G4s and DNA methylation profiles constructs a G4-DMR-DEG network, revealing nine interaction pairs. Notably, the NFATC2 promoter region sequence is confirmed to form a G4 structure, reducing promoter mCpG content and upregulating NFATC2 transcriptional activity.ConclusionsThis comprehensive study unravels the first embryonic genomic G4s landscape, highlighting the regulatory role of NFATC2 G4 in orchestrating transcriptional activity through promoter DNA methylation during myogenesis.

Human Smc5/6 recognises transcription-generated positive DNA supercoils

Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.

Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.

The mitochondrial single-stranded DNA (ssDNA)binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.

A single CAA interrupt in a DNA three-way junction containing a CAG repeat hairpin results in parity-dependent trapping.

An increasing number of human disorders are attributed to genomic expansions of short tandem repeats (STRs). Secondary DNA structures formed by STRs are believed to play an important role in expansion, while the presence of nucleotide interruptions within the pure repeat sequence isknown to delay the onset and progression of disease. We have used two single-molecule fluorescence techniques to analyse the structure and dynamics of DNA three-way junctions (3WJs) containing CAG repeat hairpin slipouts, with and without a single CAA interrupt. For a 3WJ with a (CAG)10 slipout, the CAA interrupt is preferentially located in the hairpin loop, and the branch migration dynamics are 4-fold slower than for the 3WJ with a pure (CAG)10, and 3-fold slower than a 3WJ with a pure (CAG)40 repeat. The (CAG)11 3WJ with CAA interrupt adopts a conformation that places the interrupt in or near the hairpin loop, with similar dynamics to the pure (CAG)10 and (CAG)11 3WJs. We have shown that changing a single nucleotide (G to A)in a pure repeat can have a large impact on 3WJ structure and dynamics, which may be important for the protective role of interrupts in repeat expansion diseases.

Werner helicase interacting protein 1 contributes to G-quadruplex processing in human cells

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

The Interaction of a Gemini Surfactant with a DNA Quadruplex.

DNA secondary structures are stabilized by mono- and divalent cations. To examine the stability of the DNA quadruplex formed from (TTAGGG)4, its interaction with a dicationic Gemini surfactant in standard phosphate buffer was investigated. The Gemini surfactant begins to form micelles in buffer at a cmc (critical micelle concentration) of 1.5 mM. In this study, solutions of DNA were prepared in buffer with surfactant concentrations ranging from 0.0 to 3.0 mM, i.e., above and below the cmc of the surfactant. In all samples of DNA and surfactant, a precipitate formed. The fraction of DNA precipitated depends upon both the initial DNA concentration and the initial concentration of the surfactant. In those samples where the DNA did not totally precipitate, the residual DNA assumed a quadruplex conformation. It was determined that two surfactant molecules per DNA phosphate are needed to completely precipitate all of the DNA in a particular sample. An estimated apparent K sp for the DNA:surfactant complex was determined.

Small molecule telomerase inhibitors are also potent inhibitors of telomeric C-strand synthesis.

Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.

Heme oxygenase-1 protects cells from replication stress

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress.Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation.Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

The strand exchange domain of tumor suppressor PALB2 is intrinsically disordered and promotes oligomerization-dependent DNA compaction.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.