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Current Opinion in Structural Biology Dec 2019Eukaryotic transcription factor IIH (TFIIH) is a 500 kDa-multiprotein complex that harbors two SF2-family DNA-dependent ATPase/helicase subunits and the kinase... (Review)
Review
Eukaryotic transcription factor IIH (TFIIH) is a 500 kDa-multiprotein complex that harbors two SF2-family DNA-dependent ATPase/helicase subunits and the kinase activity of Cyclin-dependent kinase 7. TFIIH serves as a general transcription factor for transcription initiation by eukaryotic RNA polymerase II and plays an important role in nucleotide excision DNA repair. Aiming to understand the molecular mechanisms of its function and regulation in two key cellular pathways, the high-resolution structure of TFIIH has been pursued for decades. Recent breakthroughs, largely enabled by methodological advances in cryo-electron microscopy, have finally revealed the structure of TFIIH and its interactions in the context of the Pol II-pre-initiation complex, and provide a first glimpse of a TFIIH-containing assembly in DNA repair. Here, we review and discuss these recent structural insights and their functional implications.
Topics: Cryoelectron Microscopy; DNA; DNA Repair; Humans; Models, Molecular; Protein Binding; Protein Conformation; Quantitative Structure-Activity Relationship; Transcription Factor TFIIH; Transcription, Genetic; Xeroderma Pigmentosum Group A Protein
PubMed: 31600675
DOI: 10.1016/j.sbi.2019.08.002 -
Proceedings of the National Academy of... Nov 2023is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb);...
is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not mutant, parasites. Comparisons with , a species that diverged from ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as -regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.
Topics: Humans; Toxoplasma; Genome; DNA, Mitochondrial; Mitochondria; Evolution, Molecular; Cell Nucleus; Sequence Analysis, DNA
PubMed: 37917792
DOI: 10.1073/pnas.2308569120 -
British Journal of Cancer Mar 2020Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for... (Review)
Review
Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for carcinogenesis. Multiple pathways have evolved to protect against both intrinsic and extrinsic genotoxic events, and recent developments have highlighted an unforeseen critical role for RNA in ensuring genome stability. It is currently unclear exactly how RNA molecules participate in the repair pathways, although many models have been proposed and it is possible that RNA acts in diverse ways to facilitate DNA repair. A number of well-documented DNA repair factors have been described to have RNA-binding capacities and, moreover, screens investigating DNA-damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair. In this review, we integrate some of these datasets to identify commonalities that might highlight novel and interesting factors for future investigations. This emerging role for RNA opens up a new dimension in the field of DNA repair; we discuss its impact on our current understanding of DNA repair processes and consider how it might influence cancer progression.
Topics: Animals; DNA; DNA Breaks, Double-Stranded; DNA Repair; Humans; RNA
PubMed: 31894141
DOI: 10.1038/s41416-019-0624-1 -
ACS Nano Feb 2021Identifying DNA species is crucial for diagnostics. For DNA identification, single-molecule DNA sequence mapping is an alternative to DNA sequencing toward fast...
Identifying DNA species is crucial for diagnostics. For DNA identification, single-molecule DNA sequence mapping is an alternative to DNA sequencing toward fast point-of-care testing, which traditionally relies on targeting and labeling DNA sequences with fluorescent labels and readout using optical imaging methods. A nanopore is a promising sensor as a complement to optical mapping with advantages of electric measurement suitable for portable devices and potential for high resolution. Here, we demonstrate a high-resolution nanopore-based DNA sequence mapping by labeling specific short sequence motifs with oligodeoxynucleotides (ODNs) using DNA methyltransferase (MTase) and detecting them using nanopores. We successfully detected ODNs down to the size of 11 nucleotides without introducing extra reporters and resolved neighboring sites with a distance of 141 bp (∼48 nm) on a single DNA molecule. To accurately locate the sequence motif positions on DNA, a nanopore data analysis method is proposed by considering DNA velocity change through nanopores and using ensemble statistics to translate the time-dependent signals to the location information. Our platform enables high-resolution detection of small labels on DNA and high-accuracy localization of them for DNA species identification in an all-electrical format. The method presents an alternative to optical techniques relying on fluorescent labels and is promising for miniature-scale integration for diagnostic applications.
Topics: Base Sequence; DNA; Nanopores; Nanotechnology; Oligodeoxyribonucleotides; Sequence Analysis, DNA
PubMed: 33478224
DOI: 10.1021/acsnano.0c07947 -
Molecular Cell Nov 2022Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required...
Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA
PubMed: 36272411
DOI: 10.1016/j.molcel.2022.09.030 -
STAR Protocols Sep 2022Our recent development of the CRAGE (chassis-independent recombinase-assisted genome engineering) system enables single-step integration of large, complex DNA constructs...
Our recent development of the CRAGE (chassis-independent recombinase-assisted genome engineering) system enables single-step integration of large, complex DNA constructs directly into bacteria genomes across multiple phyla. This protocol describes the details of the experimental design and procedures of CRAGE and extended CRAGE-Duet systems. It also describes a strategy that combines CRISPR with CRAGE, which allows implementation of CRISPR-Cas9, CRISPRa, and CRISPRi in diverse bacteria, overcoming major limitations to broaden the application of CRISPR in non-model bacterial genome engineering. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019), Wang et al. (2020), and Liu et al. (2020).
Topics: Bacteria; DNA; Genome, Bacterial; Recombinases
PubMed: 35842866
DOI: 10.1016/j.xpro.2022.101546 -
DNA Repair Sep 2019Although long overlooked, it is now well understood that DNA does not systematically assemble into a canonical double helix, known as B-DNA, throughout the entire genome... (Review)
Review
Although long overlooked, it is now well understood that DNA does not systematically assemble into a canonical double helix, known as B-DNA, throughout the entire genome but can also accommodate other structures including DNA hairpins, G-quadruplexes and RNA:DNA hybrids. Notably, these non-canonical DNA structures form preferentially at transcriptionally active loci. Acting as replication roadblocks and being targeted by multiple machineries, these structures weaken the genome and render it prone to damage, including DNA double-strand breaks (DSB). In addition, secondary structures also further accumulate upon DSB formation. Here we discuss the potential functions of pre-existing or de novo formed nucleic acid structures, as bona fide repair intermediates or repair roadblocks, especially during Transcription-Coupled DNA Double-Strand Break repair (TC-DSBR), and provide an update on the specialized protein complexes displaying the ability to remove these structures to safeguard genome integrity.
Topics: Animals; DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Eukaryota; Humans; Nucleic Acid Hybridization; RNA; Transcription, Genetic
PubMed: 31331819
DOI: 10.1016/j.dnarep.2019.102661 -
Briefings in Functional Genomics Jan 2020In silico DNA sequence generation is a powerful technology to evaluate and validate bioinformatics tools, and accordingly more than 35 DNA sequence simulation tools have... (Review)
Review
In silico DNA sequence generation is a powerful technology to evaluate and validate bioinformatics tools, and accordingly more than 35 DNA sequence simulation tools have been developed. With such a diverse array of tools to choose from, an important question is: Which tool should be used for a desired outcome? This question is largely unanswered as documentation for many of these DNA simulation tools is sparse. To address this, we performed a review of DNA sequence simulation tools developed to date and evaluated 20 state-of-art DNA sequence simulation tools on their ability to produce accurate reads based on their implemented sequence error model. We provide a succinct description of each tool and suggest which tool is most appropriate for the given different scenarios. Given the multitude of similar yet non-identical tools, researchers can use this review as a guide to inform their choice of DNA sequence simulation tool. This paves the way towards assessing existing tools in a unified framework, as well as enabling different simulation scenario analysis within the same framework.
Topics: Computer Simulation; DNA; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA; Software
PubMed: 31867604
DOI: 10.1093/bfgp/elz033 -
Biosensors Feb 2022Nucleic acids are emerging as powerful and functional biomaterials due to their molecular recognition ability, programmability, and ease of synthesis and chemical... (Review)
Review
Nucleic acids are emerging as powerful and functional biomaterials due to their molecular recognition ability, programmability, and ease of synthesis and chemical modification. Various types of nucleic acids have been used as gene regulation tools or therapeutic agents for the treatment of human diseases with genetic disorders. Nucleic acids can also be used to develop sensing platforms for detecting ions, small molecules, proteins, and cells. Their performance can be improved through integration with other organic or inorganic nanomaterials. To further enhance their biological properties, various chemically modified nucleic acid analogues can be generated by modifying their phosphodiester backbone, sugar moiety, nucleobase, or combined sites. Alternatively, using nucleic acids as building blocks for self-assembly of highly ordered nanostructures would enhance their biological stability and cellular uptake efficiency. In this review, we will focus on the development and biomedical applications of structural and functional natural nucleic acids, as well as the chemically modified nucleic acid analogues over the past ten years. The recent progress in the development of functional nanomaterials based on self-assembled DNA-based platforms for gene regulation, biosensing, drug delivery, and therapy will also be presented. We will then summarize with a discussion on the advanced development of nucleic acid research, highlight some of the challenges faced and propose suggestions for further improvement.
Topics: Biosensing Techniques; DNA; Drug Delivery Systems; Humans; Nanostructures; Nanotechnology; Nucleic Acids
PubMed: 35200353
DOI: 10.3390/bios12020093 -
Molecular Cell May 2022Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with... (Review)
Review
Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.
Topics: Biophysics; DNA; High-Throughput Nucleotide Sequencing; Molecular Biology; Sequence Analysis, DNA
PubMed: 35561688
DOI: 10.1016/j.molcel.2022.04.024