scholarly journals Dimer–dimer stacking interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

2010 ◽  
Vol 427 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Clare Jelinska ◽  
Biljana Petrovic-Stojanovska ◽  
W. John Ingledew ◽  
Malcolm F. White
Keyword(s):  
Stacking Interactions ◽  
Nucleic Acid Binding ◽  
Dimer Interface ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Helical Protein ◽  
End To End ◽  
Polypeptide Backbone

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer–dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg59 and Phe60. In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe60 in forming the dimer–dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer–dimer interaction forms in the presence of dsDNA.

scholarly journals RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

2022 ◽  
Author(s):  
Shan Qi ◽  
Javier Mota ◽  
Siu-Hong Chan ◽  
Johanna Villarreal ◽  
Nan Dai ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rna Binding ◽  
Methyl Group ◽  
High Affinity ◽  
Methylation Of Dna ◽  
Dna And Rna ◽  
Close Proximity ◽  
Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

scholarly journals Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD

2018 ◽  
Vol 29 (4) ◽  
pp. 1165-1181 ◽  
Author(s):  
Takanori Komada ◽  
Hyunjae Chung ◽  
Arthur Lau ◽  
Jaye M. Platnich ◽  
Paul L. Beck ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Inflammasome Activation ◽  
Dna Uptake ◽  
Chronic Injury ◽  
Caspase 1 ◽  
Double Stranded Dna ◽  
Molecular Pattern ◽  
Macrophage Uptake

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1β cleavage in Aim2−/− or WT mice that received WT bone marrow than in WT mice that received Aim2−/− bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5–6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1β, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1β levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

scholarly journals Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2

2020 ◽  
Vol 48 (7) ◽  
pp. 3869-3887 ◽  
Author(s):  
Linlin Hou ◽  
Yuanjie Wei ◽  
Yingying Lin ◽  
Xiwei Wang ◽  
Yiwei Lai ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Mobility ◽  
Binding Motif ◽  
Dna And Rna ◽  
Somatic Cell Reprogramming ◽  
Double Stranded Dna

Abstract Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

scholarly journals Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 253 ◽  
Author(s):  
Mauricio Comas-Garcia
Keyword(s):  
Rna Viruses ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Dna And Rna ◽  
Relative Contribution ◽  
Double Stranded Dna ◽  
Positive Sense ◽  
Infectious Cycle

The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.

scholarly journals Intrusion of a DNA Repair Protein in the RNome World: Is This the Beginning of a New Era?

2009 ◽  
Vol 30 (2) ◽  
pp. 366-371 ◽  
Author(s):  
Gianluca Tell ◽  
David M. Wilson ◽  
Chow H. Lee
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Mrna Level ◽  
Rna Metabolism ◽  
Coding Region ◽  
Control Of Gene Expression ◽  
Dna And Rna ◽  
New Findings

ABSTRACT Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is known to be involved in base excision DNA repair, acting as the major abasic endonuclease; the protein also functions as a redox coactivator of several transcription factors that regulate gene expression. Recent findings highlight a novel role for APE1 in RNA metabolism. The new findings are as follows: (i) APE1 interacts with rRNA and ribosome processing protein NPM1 within the nucleolus; (ii) APE1 interacts with proteins involved in ribosome assembly (i.e., RLA0, RSSA) and RNA maturation (i.e., PRP19, MEP50) within the cytoplasm; (iii) APE1 cleaves abasic RNA; and (iv) APE1 cleaves a specific coding region of c-myc mRNA in vitro and influences c-myc mRNA level and half-life in cells. Such findings on the role of APE1 in the posttranscriptional control of gene expression could explain its ability to influence diverse biological processes and its relocalization to cytoplasmic compartments in some tissues and tumors. In addition, we propose that APE1 serves as a “cleansing” factor for oxidatively damaged abasic RNA, establishing a novel connection between DNA and RNA surveillance mechanisms. In this review, we introduce questions and speculations concerning the role of APE1 in RNA metabolism and discuss the implications of these findings in a broader evolutionary context.

scholarly journals Investigating site-selection mechanisms of retroviral integration in supercoiled DNA braids

2021 ◽  
Vol 18 (181) ◽  
pp. 20210229
Author(s):  
G. Forte ◽  
D. Michieletto ◽  
D. Marenduzzo ◽  
E. Orlandini
Keyword(s):  
Mechanistic Explanation ◽  
Bending Energy ◽  
Retroviral Integration ◽  
Double Stranded Dna ◽  
Integration Sites ◽  
Set Up ◽  
Selection Mechanisms ◽  
Temporally Correlated

We theoretically study the integration of short viral DNA in a DNA braid made up by two entwined double-stranded DNA molecules. We show that the statistics of single integration events substantially differ in the straight and buckled, or plectonemic, phase of the braid and are more likely in the latter. We further discover that integration is most likely close to plectoneme tips, where the larger bending energy helps overcome the associated energy barrier and that successive integration events are spatio-temporally correlated, suggesting a potential mechanistic explanation of clustered integration sites in host genomes. The braid geometry we consider provides a novel experimental set-up to quantify integration in a supercoiled substrate in vitro , and to better understand the role of double-stranded DNA topology during this process.

scholarly journals Hnrnpul1 loss of function affects skeletal and limb development

2020 ◽  
Author(s):  
Danielle L Blackwell ◽  
Sherri D Fraser ◽  
Oana Caluseriu ◽  
Claudia Vivori ◽  
Paul MK Gordon ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Limb Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Dna And Rna ◽  
Developmental Role

AbstractMutations in RNA binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb and neurological symptoms. Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. Here, we show a developmental role for hnrnpul1 in zebrafish fin and craniofacial development, and in adult onset scoliosis. Furthermore, we demonstrate a role of hnrnpul1 in alternative splicing regulation. In two siblings with congenital limb malformations, whole exome sequencing detected a frameshift variant in HNRNPUL1; the developmental role of this gene in humans has not been explored. Our data suggest an important developmental role of hnRNPUL1 in both zebrafish and humans. Although there are differences in phenotypes between species, our data suggests potential conservation of ancient regulatory circuits involving hnRNPUL1 in these phylogenetically distant species.Summary statementA zebrafish model of loss of Hnrnpul1 shows alternative splicing defects and results in limb growth, craniofacial tendon, and skeletal anomalies.

scholarly journals The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

1998 ◽  
Vol 18 (6) ◽  
pp. 3580-3585 ◽  
Author(s):  
Wenjin Zheng ◽  
Stephen Albert Johnston
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Dual Function ◽  
Nucleic Acid Binding ◽  
Peptide Cleavage ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Bleomycin Hydrolase ◽  
Nucleic Acid Binding Activity ◽  
Rna And Dna

ABSTRACT Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked double-stranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.

scholarly journals Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro

Science ◽  
2020 ◽  
Vol 370 (6513) ◽  
pp. eabc8420 ◽  
Author(s):  
Devin E. Christensen ◽  
Barbie K. Ganser-Pornillos ◽  
Jarrod S. Johnson ◽  
Owen Pornillos ◽  
Wesley I. Sundquist
Keyword(s):  
Cell Extract ◽  
Free System ◽  
Double Stranded Dna ◽  
A Cell ◽  
Dna Ends ◽  
Hiv 1 ◽  
Target Plasmid

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract–dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

scholarly journals Role of the C Terminus of Foamy Virus Gag in RNA Packaging and Pol Expression

2004 ◽  
Vol 78 (17) ◽  
pp. 9423-9430 ◽  
Author(s):  
Carolyn R. Stenbak ◽  
Maxine L. Linial
Keyword(s):  
Foamy Virus ◽  
Nucleic Acid Binding ◽  
Rna Packaging ◽  
Binding Properties ◽  
Gag Protein ◽  
C Terminus ◽  
Foamy Viruses ◽  
The Matrix

ABSTRACT Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.

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