DNA Tracts Research Articles

The RNA-binding protein PCBP1 modulates transcription by recruiting the G-quadruplex-specific helicase DHX9

PCBP1, polycytosine (poly(C)) binding protein 1, an RNA and single-stranded DNA (ssDNA) binding protein, binds poly(C) DNA tracts but it remains unclear whether its ability to bind ssDNA contributes to transcriptional regulation. Here, we report that PCBP1's DNA binding sites are enriched at transcription start sites and that by binding to promoter regions, PCBP1 regulates transcription in addition to splicing and translation. At PCBP1 target genes, we show that PCBP1 interacts with several RNA/DNA hybrid (R-loop) associated G-quadruplex resolving helicases. Furthermore, we find that PCBP1 interacts with RNA Helicase A (DHX9) to modulate transcription by regulating DHX9 accumulation and activity. PCBP1 depletion leads to defects in R-loop processing and dysregulation of transcription of PCBP1 target genes. PCBP1's high sequence specificity and interaction with helicases suggest that its mechanism in transcription involves guiding helicases to specific loci during transcription, thereby modulating their activity.

CORRECTING MODEL MISSPECIFICATION IN RELATIONSHIP ESTIMATES.

The datasets of large genotyping biobanks and direct-to-consumer genetic testing companies contain many related individuals. Until now, it has been widely accepted that the most distant relationships that can be detected are around fifteen degrees (approximately 8 th cousins) and that practical relationship estimates have a ceiling around ten degrees (approximately 5 th cousins). However, we show that these assumptions are incorrect and that they are due to a misapplication of relationship estimators. In particular, relationship estimators are applied almost exclusively to putative relatives who have been identified because they share detectable tracts of DNA identically by descent (IBD). However, no existing relationship estimator conditions on the event that two individuals share at least one detectable segment of IBD anywhere in the genome. As a result, the relationship estimates obtained using existing estimators are dramatically biased for distant relationships, inferring all sufficiently distant relationships to be around ten degrees regardless of the depth of the true relationship. Existing relationship estimators are derived under a model that assumes that each pair of related individuals shares a single common ancestor (or mating pair of ancestors). This model breaks down for relationships beyond 10 generations in the past because individuals share many thousands of cryptic common ancestors due to pedigree collapse. We first derive a corrected likelihood that conditions on the event that at least one segment is observed between a pair of putative relatives and we demonstrate that the corrected likelihood largely eliminates the bias in estimates of pairwise relationships and provides a more accurate characterization of the uncertainty in these estimates. We then reformulate the relationship inference problem to account for the fact that individuals share many common ancestors, not just one. We demonstrate that the most distant relationship that can be inferred using IBD may be 200 degrees or more, rather than ten, extending the time-to-common ancestor from approximately 300 years in the past to approximately 3,000 years in the past or more. This dramatic increase in the range of relationship estimators makes it possible to infer relationships whose common ancestors lived before historical events such as European settlement of the Americas, the Transatlantic Slave Trade, and the rise and fall of the Roman Empire.

Horizontally transferred mitochondrial DNA tracts become circular by microhomology-mediated repair pathways.

The holoparasitic plant Lophophytum mirabile exhibits remarkable levels of mitochondrial horizontal gene transfer (HGT). Gathering comparative data from other individuals and host plants can provide insights into the HGT process. We sequenced the mitochondrial genome (mtDNA) from individuals of two species of Lophophytum and from mimosoid hosts. We applied a stringent phylogenomic approach to elucidate the origin of the whole mtDNAs, estimate the timing of the transfers, and understand the molecular mechanisms involved. Ancestral and recent HGT events replaced and enlarged the multichromosomal mtDNA of Lophophytum spp., with the foreign DNA ascending to 74%. A total of 14 foreign mitochondrial chromosomes originated from continuous regions in the host mtDNA flanked by short direct repeats. These foreign tracts are circularized by microhomology-mediated repair pathways and replicate independently until they are lost or they eventually recombine with other chromosomes. The foreign noncoding chromosomes are variably present in the population and likely evolve by genetic drift. We present the 'circle-mediated HGT' model in which foreign mitochondrial DNA tracts become circular and are maintained as plasmid-like molecules. This model challenges the conventional belief that foreign DNA must be integrated into the recipient genome for successful HGT.

Origins of lineage-specific elements via gene duplication, relocation, and regional rearrangement in Neurospora crassa.

The origin of new genes has long been a central interest of evolutionary biologists. However, their novelty means that they evade reconstruction by the classical tools of evolutionary modelling. This evasion of deep ancestral investigation necessitates intensive study of model species within well-sampled, recently diversified, clades. One such clade is the model genus Neurospora, members of which lack recent gene duplications. Several Neurospora species are comprehensively characterized organisms apt for studying the evolution of lineage-specific genes (LSGs). Using gene synteny, we documented that 78% of Neurospora LSG clusters are located adjacent to the telomeres featuring extensive tracts of non-coding DNA and duplicated genes. Here, we report several instances of LSGs that are likely from regional rearrangements and potentially from gene rebirth. To broadly investigate the functions of LSGs, we assembled transcriptomics data from 68 experimental data points and identified co-regulatory modules using Weighted Gene Correlation Network Analysis, revealing that LSGs are widely but peripherally involved in known regulatory machinery for diverse functions. The ancestral status of the LSG mas-1, a gene with roles in cell-wall integrity and cellular sensitivity to antifungal toxins, was investigated in detail alongside its genomic neighbours, indicating that it arose from an ancient lysophospholipase precursor that is ubiquitous in lineages of the Sordariomycetes. Our discoveries illuminate a "rummage region" in the N. crassa genome that enables the formation of new genes and functions to arise via gene duplication and relocation, followed by fast mutation and recombination facilitated by sequence repeats and unconstrained non-coding sequences.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances invitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Abstract 3098: Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core

Abstract Quadruplex nucleic acids (G4s) are four-stranded arrangements formed by guanine-rich tracts of DNA or RNA, held together by Hoogsteen hydrogen-bonded guanine quartets. G4 sequences are widely though not randomly distributed in the human genome. They are over-represented in telomeres and in the regulatory regions of many cancer-related genes such as KIT, MYC, VEGF, RAS and HIF. G4 structures can be unwound by helicase activity but can be stabilized by the binding of high-affinity small molecules. The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative, has nanomolar affinity and selectivity for several DNA and RNA G4s over duplex DNA. QN-302 has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It has significant anti-tumor activity in several models for PDAC - in a MIA-PACA2 xenograft, in the genetic KPC model and in an orthotopic BX-PC3 model. QN-302 is currently in an advanced stage of pre-IND development by Qualigen Inc as a novel anti-cancer agent. QN-302 is the latest in several generations of NDs developed by us and designed by computer modelling based on earlier G4 co-crystal structures determined in this laboratory. The database of seven ND-containing crystal structures includes four different NDs with distinct side chain end groups, all with intramolecular telomeric parallel G4s. End-groups include N-methyl piperazine, hydroxyl, morpholino and N-dimethyl. These structures have been systematically compared for example using least-squares overlays of the structures. All the structures have their four substituents positioned each in a G4 groove, although there are significant differences in orientations of the end groups. Surprisingly, the naphthalene diimide cores do not retain the same orientation in each structure, with three distinct orientations being apparent. Examination of ND core overlap with the adjacent G-quartet has revealed variation in the degree of heterocycle-guanine base overlap, which is dependent on the nature and size of each substituent. However, all structures show minimal ND-guanine overlap, with typically just one out of four guanines in a G-quartet being involved. This strongly implies that most of the contribution to ND-G4 affinity comes from the side chains, and especially from the protonated end groups. These and other observations have led us to develop a structure-activity framework for tetra-substituted NDs, which has culminated in their optimization and the design of QN-302, with enhanced cellular potency and G4 affinity compared to previous NDs. Citation Format: Stephen Neidle. Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3098.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT)

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Recent Microscopy Advances and the Applications to Huntington's Disease Research.

Huntingtin is a 3144 amino acid protein defined as a scaffold protein with many intracellular locations that suggest functions in these compartments. Expansion of the CAG DNA tract in the huntingtin first exon is the cause of Huntington’s disease. An important tool in understanding the biological functions of huntingtin is molecular imaging at the single-cell level by microscopy and nanoscopy. The evolution of these technologies has accelerated since the Nobel Prize in Chemistry was awarded in 2014 for super-resolution nanoscopy. We are in a new era of light imaging at the single-cell level, not just for protein location, but also for protein conformation and biochemical function. Large-scale microscopy-based screening is also being accelerated by a coincident development of machine-based learning that offers a framework for truly unbiased data acquisition and analysis at very large scales. This review will summarize the newest technologies in light, electron, and atomic force microscopy in the context of unique challenges with huntingtin cell biology and biochemistry.

Heterogeneous migration routes of DNA triplet repeat slip-outs

It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.

Diploid-associated adaptation to chronic low-dose UV irradiation requires homologous recombination in Saccharomyces cerevisiae.

Ultraviolet-induced DNA lesions impede DNA replication and transcription and are therefore a potential source of genome instability. Here, we performed serial transfer experiments on nucleotide excision repair-deficient (rad14Δ) yeast cells in the presence of chronic low-dose ultraviolet irradiation, focusing on the mechanisms underlying adaptive responses to chronic low-dose ultraviolet irradiation. Our results show that the entire haploid rad14Δ population rapidly becomes diploid during chronic low-dose ultraviolet exposure, and the evolved diploid rad14Δ cells were more chronic low-dose ultraviolet-resistant than haploid cells. Strikingly, single-stranded DNA, but not pyrimidine dimer, accumulation is associated with diploid-dependent fitness in response to chronic low-dose ultraviolet stress, suggesting that efficient repair of single-stranded DNA tracts is beneficial for chronic low-dose ultraviolet tolerance. Consistent with this hypothesis, homologous recombination is essential for the rapid evolutionary adaptation of diploidy, and rad14Δ cells lacking Rad51 recombinase, a key player in homologous recombination, exhibited abnormal cell morphology characterized by multiple RPA-yellow fluorescent protein foci after chronic low-dose ultraviolet exposure. Furthermore, interhomolog recombination is increased in chronic low-dose ultraviolet-exposed rad14Δ diploids, which causes frequent loss of heterozygosity. Thus, our results highlight the importance of homologous recombination in the survival and genomic stability of cells with unrepaired lesions.

Proteinopathies associated to repeat expansion disorders.

The most common neurodegenerative disorders, such as Alzheimer's or Parkinson's diseases, are characterized by synaptic dysfunction, neuronal loss and proteinaceous aggregates in central nervous system. The deposition of misfolded proteins constitutes neuropathological hallmarks of these diseases, grouped in the generic term of proteinopathies. Apart from these, other neurodegenerative diseases are characterized by genetic abnormalities like unstable repetitive simple sequence tracts (microsatellites) dispersed throughout the human genome. They are called repeat expansion disorders and include, for example, Huntington's disease or frontotemporal dementia/amyotrophic lateral sclerosis phenotypes associated to an expansion in C9ORF72. The presence of the expanded DNA tract leads to molecular alterations at the DNA, RNA and protein levels associated to distinct mechanisms, such as loss-of-function (LOF), gain-of-function (GOF) including misfolding of physiological or mutant proteins, favoring their polymerization and aggregation. Therefore, specific proteinopathies also arise from these repeat expansion disorders. The molecular description of the nature and location of expanded tracts, highlighting the consequences onto clinical phenotypes will be first described. Specific focuses on the three pathomechanisms of the repeat expansions associated to proteinopathies will then be addressed. Lastly, we will show how progress in the understanding of these different mechanisms has led to recent advances in new/innovative therapeutic approaches and emergence of associated biomarkers.

Method for identification of 10 SSR markers from monkey genomes and its statistical inference with One & Two-way ANOVA

DNA tracts that include simple sequence repeats (SSRs), sometimes known as genetic "stutters), are composed of a few to many tandem repetitions of a short base-pair motif. These sequences frequently mutate, changing the amount of repetitions. SSRs are frequently found in promoters, untranslated regions, and even coding sequences, therefore these alterations can significantly affect practically every aspect of gene activity. SSR alleles can also contribute to normal diversity in brain and behavioural features. Mutational expansion of certain triplet repeats is the cause of a number of inherited neurodegenerative diseases. Due to its importance in genetic research, in this paper we explored Ten SSR markers TAGA, TCAT, GAAT, AGAT, AGAA, GATA, TATC, CTTT, TCTG and TCTA that are identified from the genomes of Eleven distinct monkeys: A.Nancymaae, C.C.Imitator, C.Atys, M.Leucophaeus, P.Paniscus, R.Bieti, R.Roxellana, S.Boliviensis, T.Syrichta, C.A.Palliatus and M.Nemestrina using pattern matching mechanism. We identified 4bp SSR from eleven monkey dataset's Unchr chromosome mainly in this paper. The proposed approach finds the exact place/location of the SSR's and number of times that it appears in the given genome sequence. The identified patterns are analyzed with One-way and Two-way ANOVA that gives better analysis which is useful for genomic studies. Also, this 4bp Ten SSR markers data is a valuable to illustrate genetic variation of genomic study.•The great specificity of data sets produced from monkey genomes with pattern matching has been demonstrated.•These findings show that SSR identification could be a useful tool for determining genome similarity and comparability.•Researchers can use the raw sequencing data to conduct additional bioinformatics analysis.

Microsatellite Instability Analysis (MSA) for Bladder Cancer: Past History and Future Directions.

Microsatellite instability (MSI), the spontaneous loss or gain of nucleotides from repetitive DNA tracts, is a diagnostic phenotype for gastrointestinal, endometrial, colorectal, and bladder cancers; yet a landscape of instability events across a wider variety of cancer types is beginning to be discovered. The epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches to the detection of the MSI phenotype of tumors using NGS have been developed. Bladder cancer (here we refer to transitional carcinoma of the bladder) is a major cause of morbidity and mortality in the Western world. Cystoscopy, a gold standard for the detection of bladder cancer, is invasive and sometimes carries unwanted complications, while its cost is relatively high. Urine cytology is of limited value due to its low sensitivity, particularly to low-grade tumors. Therefore, over the last two decades, several new “molecular assays” for the diagnosis of urothelial cancer have been developed. Here, we provide an update on the development of a microsatellite instability assay (MSA) and the development of MSA associated with bladder cancers, focusing on findings obtained from urine analysis from bladder cancer patients as compared with individuals without bladder cancer. In our review, based on over 18 publications with approximately 900 sample cohorts, we provide the sensitivity (87% to 90%) and specificity (94% to 98%) of MSA. We also provide a comparative analysis between MSA and other assays, as well as discussing the details of four different FDA-approved assays. We conclude that MSA is a potentially powerful test for bladder cancer detection and may improve the quality of life of bladder cancer patients.

Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

5-Aza-7-deazaguanine-Isoguanine and Guanine-Isoguanine Base Pairs in Watson-Crick DNA: The Impact of Purine Tracts, Clickable Dendritic Side Chains, and Pyrene Adducts.

The Watson–Crick coding system depends on the molecular recognition of complementary purine and pyrimidine bases. Now, the construction of hybrid DNAs with Watson–Crick and purine–purine base pairs decorated with dendritic side chains was performed. Oligonucleotides with single and multiple incorporations of 5‐aza‐7‐deaza‐2′‐deoxyguanosine, its tripropargylamine derivative, and 2′‐deoxyisoguanosine were synthesized. Duplex stability decreased if single modified purine–purine base pairs were inserted, but increased if pyrene residues were introduced by click chemistry. A growing number of consecutive 5‐aza‐7‐deazaguanine–isoguanine base pairs led to strong stepwise duplex stabilization, a phenomenon not observed for the guanine–isoguanine base pair. Spacious residues are well accommodated in the large groove of purine–purine DNA tracts. Changes to the global helical structure monitored by circular dichroism spectroscopy show the impact of functionalization to the global double‐helix structure. This study explores new areas of molecular recognition realized by purine base pairs that are complementary in hydrogen bonding, but not in size, relative to canonical pairs.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells

AbstractFanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2−/−SLFN11−/− HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2−/− cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2−/− cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

Genome-wide detection of short tandem repeat expansions by long-read sequencing

BackgroundShort tandem repeat (STR), or “microsatellite”, is a tract of DNA in which a specific motif (typically < 10 base pairs) is repeated multiple times. STRs are abundant throughout the human genome, and specific repeat expansions may be associated with human diseases. Long-read sequencing coupled with bioinformatics tools enables the estimation of repeat counts for STRs. However, with the exception of a few well-known disease-relevant STRs, normal ranges of repeat counts for most STRs in human populations are not well known, preventing the prioritization of STRs that may be associated with human diseases.ResultsIn this study, we extend a computational tool RepeatHMM to infer normal ranges of 432,604 STRs using 21 long-read sequencing datasets on human genomes, and build a genomic-scale database called RepeatHMM-DB with normal repeat ranges for these STRs. Evaluation on 13 well-known repeats show that the inferred repeat ranges provide good estimation to repeat ranges reported in literature from population-scale studies. This database, together with a repeat expansion estimation tool such as RepeatHMM, enables genomic-scale scanning of repeat regions in newly sequenced genomes to identify disease-relevant repeat expansions. As a case study of using RepeatHMM-DB, we evaluate the CAG repeats of ATXN3 for 20 patients with spinocerebellar ataxia type 3 (SCA3) and 5 unaffected individuals, and correctly classify each individual.ConclusionsIn summary, RepeatHMM-DB can facilitate prioritization and identification of disease-relevant STRs from whole-genome long-read sequencing data on patients with undiagnosed diseases. RepeatHMM-DB is incorporated into RepeatHMM and is available at https://github.com/WGLab/RepeatHMM.

Observations of, and Insights into, Cystic Fibrosis Mucus Heterogeneity in the Pre-Modulator Era: Sputum Characteristics, DNA and Glycoprotein Content, and Solubilization Time

Airway obstruction with chronic inflammation and infection are major contributors to the lung damage and mortality of cystic fibrosis (CF). A better understanding of the congested milieu of CF airways will aid in improving therapeutic strategies. This article retrospectively reports our observations, and discusses insights gained in the handling and analysis of CF sputa. CF and non-CF mucus samples were surveyed for morphological features by electron microscopy and analyzed for the macromolecular dry weight (MDW), total protein, lipid, carbohydrate, and DNA. Mucus character was investigated with chemical solubilization time as a comparative tool. CF mucus appeared distinctly thick, viscous, and heterogeneous, with neutrophils as the dominant immune cell. CF sputum DNA content varied markedly for and between individuals (~1–10% MDW), as did solubilization times (~1–20 h). CF Sputum DNA up to 7.1% MDW correlated positively with solubilization time, whereas DNA &gt;7.1% MDW correlated negatively. 3D analysis of CF sputa DNA, GP, and solubilization times revealed a dynamic and predictive relationship. Reflecting on the heterogeneous content and character of CF mucus, and the possible interplay in space and time in the respiratory tract of polymeric DNA and mucous glycoproteins, we highlight it’s potential to affect infection-related airway pathologies and the success of therapeutic interventions.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.