Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “DNA”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

DNA damage in internal organs after cutaneous exposure to sulphur mustard

Sulphur mustard (SM) is a chemical warfare agent that attacks mainly skin, eye and lungs. Due to its lipophilic properties, SM is also able to diffuse through the skin and reach internal organs. DNA represents one of the most critical molecular targets of this powerful alkylating agent which modifies DNA structure by forming monoadducts and biadducts. These DNA lesions are involved in the acute toxicity of SM as well as its long-term carcinogenicity. In the present work we studied the formation and persistence of guanine and adenine monoadducts and guanine biadducts in the DNA of brain, lungs, kidneys, spleen, and liver of SKH-1 mice cutaneously exposed to 2, 6 and 60 mg/kg of SM. SM-DNA adducts were detected in all studied organs, except in liver at the two lowest doses. Brain and lungs were the organs with the highest level of SM-DNA adducts, followed by kidney, spleen and liver. Monitoring the level of adducts for three weeks after cutaneous exposure showed that the lifetime of adducts were not the same in all organs, lungs being the organ with the longest persistence. Diffusion from skin to internal organs was much more efficient at the highest compared to the lowest dose investigated as the result of the loss of the skin barrier function. These data provide novel information on the distribution of SM in tissues following cutaneous exposures and indicate that brain is an important target. - Highlights: • Sulphur mustard reaches internal organs after skin exposure • Adducts are detected in the DNA of internal organs • Brain is the organ with the highest level of DNA damage • The barrier function of skin is lost at high dose of sulphur mustard • DNA adducts persist in organs for 2 or 3 weeks.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Regulation of the gyr operon of Mycobacterium tuberculosis by overlapping promoters, DNA topology, and reiterative transcription

Highlights: • Demonstrated reiterative transcription at the gyr overlapping promoters in Mycobacterium tuberculosis. • Regulation by reiterative transcription is DNA topology-dependent. • Another player in the regulation of DNA Gyrase. DNA gyrase introduces negative supercoils into DNA to maintain topological homeostasis. The genes encoding gyrase, gyrB and gyrA, form a dicistronic operon in Mycobacterium tuberculosis (Mtb) and other actinobacteria. Earlier work indicated that DNA relaxation stimulates the expression of the gyr genes, a phenomenon termed relaxation-stimulated transcription (RST). The present study addresses the underlying mechanism of gyr operon regulation. The operon is regulated by overlapping and divergently oriented promoters located upstream of gyrB. The principal promoter, P{sub gyrB1}, drives transcription of the operon, while a weak “reverse” promoter, P{sub gyrR}, transcribes in opposite direction. We demonstrate that P{sub gyrR} plays a role in fine tuning gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not found in Mtb. In vitro transcription assays showed that RT at P{sub gyrR} depended on the negatively supercoiled state of the DNA template. The principal promoter, P{sub gyrB1}, was also sensitive to DNA supercoiling, but it was stimulated by DNA relaxation. Moreover, RNA polymerase binding to the promoter was efficient at P{sub gyrB1} when template DNA was relaxed, whereas binding to P{sub gyrR} was preferred when DNA was supercoiled. Thus, a collaboration between RST and RT governs the regulation of the gyr operon; the differing sensitivity of the two overlapping promoters to superhelix density explains how gyrase expression responds to changes in supercoiling to determine the efficiency of transcription initiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Significant accumulation of persistent organic pollutants and dysregulation in multiple DNA damage repair pathways in the electronic-waste-exposed populations

Electronic waste (e-waste) has created a worldwide environmental and health problem, by generating a diverse group of hazardous compounds such as persistent organic pollutants (POPs). Our previous studies demonstrated that populations from e-waste exposed region have a significantly higher level of chromosomal aberrancy and incidence of DNA damage. In this study, we further demonstrated that various POPs persisted at a significantly higher concentration in the exposed group than those in the unexposed group. The level of reactive oxygen species and micronucleus rate were also significantly elevated in the exposed group. RNA sequencing analysis revealed 31 genes in DNA damage responses and repair pathways that were differentially expressed between the two groups (Log 2 ratio >1 or <−1). Our data demonstrated that both females and males of the exposed group have activated a series of DNA damage response genes; however many important DNA repair pathways have been dysregulated. Expressions of NEIL1/3 and RPA3, which are critical in initiating base pair and nucleotide excision repairs respectively, have been downregulated in both females and males of the exposed group. In contrast, expression of RNF8, an E3 ligase involved in an error prone non-homologous end joining repair for DNA double strand break, was upregulated in both genders of the exposed group. The other genes appeared to be differentially expressed only when the males or females of the two groups were compared respectively. Importantly, the expression of cell cycle regulatory gene CDC25A that has been implicated in multiple kinds of malignant transformation was significantly upregulated among the exposed males while downregulated among the exposed females. In conclusion, our studies have demonstrated significant correlations between e-waste disposing and POPs accumulation, DNA lesions and dysregulation of multiple DNA damage repair mechanisms in the residents of the e-waste exposed region. - Highlights: • We compared concentration of POPs, ROS and micronucleus rate in POPs exposed area. • Significant accumulation of POPs homologous in the e-waste exposed residents. • DNA damage and DNA damage repair pathways have been differentially activated. • Females and males in the exposed group have different responses to the DNA damage. • Exposed males may be more prone to undergo malignant transformation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2015

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes might be destroyed. In conclusion, we believe that the continued rapid evolution of CRISPR/Cas technology will soon have a major, possibly revolutionary, impact on the field of virology. - Highlights: • Bacterial CRISPR/Cas systems can edit specific DNA sequences in mammalian cells. • CRISPR/Cas systems could eliminate latent or persistent DNA viruses in vivo. • CRISPR/Cas could also be used to screen for viral co-factors or restriction factors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI

Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein–DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

DNA origami protection and molecular interfacing through engineered sequence-defined peptoids

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson−Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1–9) with two types of architectures, termed as “brush” and “block,” were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

36 MATERIALS SCIENCE↗
DOE OSTI2015

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

BuD, a helix–loop–helix DNA-binding domain for genome modification

Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2016

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. - Highlights: • PM exposure induces DNA damage repair gene expression. • Inhibition of ATM prevented PM-induced follicle depletion. • PKCδ deficiency did not impact PM-induced ovotoxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA’s structure-specific DNA unwinding properties and impairs its activity in vivo. Here, our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Swelling Inhibition of Liquid Crystalline Colloidal Montmorillonite and Beidellite Clays by DNA

Exploring the interaction of nucleic acids with clay minerals is important to understand such issues as the persistence in soils of biomolecules and the appearance of genetic polymers in prebiotic environments. Colloidal dispersions of double stranded DNA and clay nanosheets may also provide interesting model systems to study the statistical physics of mixtures of semi-flexible rods and plates. Here, we show that adding very small amounts of DNA to liquid-crystalline montmorillonite and beidellite smectite clay suspensions strongly widens the isotropic/nematic phase coexistence region. Moreover, a spectroscopic study shows that, upon DNA addition, the first DNA molecules adsorb onto the clay particles. Remarkably, synchrotron small-angle X-ray scattering experiments reveal that the average distance between the clay sheets, in the nematic phase at coexistence, decreases with increasing DNA concentration and that the inhibition of swelling by DNA becomes almost independent of clay concentration. We interpret this DNA-mediated attraction between clay nanosheets by bridging conformations of DNA strands (plates on a string structure). In addition to bridging, DNA chains can form "loops" between sections adsorbed on the same particle, giving rise to sheet repulsions due to protruding loops. Furthermore, this interpretation agrees with the observed inter-clay spacings being dependent only on the DNA concentration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling

Background & Aims: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. Methods: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA 57-80 ), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. Results: The TcdA 57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA 57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. Conclusions: TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a–RNA–DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cryo-EM structure of AAV2 Rep68 bound to integration site AAVS1: insights into the mechanism of DNA melting

Abstract The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPγS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process.

Jaiswal, Rahul↗
DOE OSTI2017

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Role for first zinc finger of WT1 in DNA sequence specificity: Denys–Drash syndrome-associated WT1 mutant in ZF1 enhances affinity for a subset of WT1 binding sites

Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1–4). An alternative splicing of Wt1 can add three additional amino acids—lysine (K), threonine (T) and serine (S)—between ZF3 and ZF4. In the –KTS isoform, ZF2–4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type –KTS isoform ZF1–4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2–4, ZF1 can be positioned continuously in the DNA major groove forming a ‘near-cognate’ complex. Second, while ZF2–4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys–Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.

59 BASIC BIOLOGICAL SCIENCES↗