Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Nat Commun ; 14(1): 7072, 2023 11 03.
Article in English | MEDLINE | ID: mdl-37923737

ABSTRACT

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.


Subject(s)
DNA, Viral , Spumavirus , DNA, Viral/metabolism , Integrases/metabolism , Retroviridae/genetics , Retroviridae/metabolism , Spumavirus/genetics , Spumavirus/metabolism , DNA, Complementary , Virus Integration
2.
Nat Rev Genet ; 24(12): 816-833, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37380761

ABSTRACT

Ever since microRNAs (miRNAs) were first recognized as an extensive gene family >20 years ago, a broad community of researchers was drawn to investigate the universe of small regulatory RNAs. Although core features of miRNA biogenesis and function were revealed early on, recent years continue to uncover fundamental information on the structural and molecular dynamics of core miRNA machinery, how miRNA substrates and targets are selected from the transcriptome, new avenues for multilevel regulation of miRNA biogenesis and mechanisms for miRNA turnover. Many of these latest insights were enabled by recent technological advances, including massively parallel assays, cryogenic electron microscopy, single-molecule imaging and CRISPR-Cas9 screening. Here, we summarize the current understanding of miRNA biogenesis, function and regulation, and outline challenges to address in the future.


Subject(s)
MicroRNAs , MicroRNAs/genetics , Transcriptome
3.
J Biol Chem ; 296: 100550, 2021.
Article in English | MEDLINE | ID: mdl-33744295

ABSTRACT

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.


Subject(s)
Histones/metabolism , Integrases/metabolism , Nucleosomes/metabolism , Spumavirus/metabolism , Viral Proteins/metabolism , Virus Integration , Catalytic Domain , Chromatin/genetics , Chromatin/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Genome, Viral , Humans , Integrases/genetics , Protein Multimerization , Spumavirus/genetics , Spumavirus/growth & development , Viral Proteins/chemistry , Viral Proteins/genetics
4.
J Vis Exp ; (153)2019 11 08.
Article in English | MEDLINE | ID: mdl-31762446

ABSTRACT

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.


Subject(s)
Oxygen/metabolism , Protocatechuate-3,4-Dioxygenase/isolation & purification , Protocatechuate-3,4-Dioxygenase/metabolism , Photobleaching , Pseudomonas putida/enzymology , Substrate Specificity
5.
Sci Rep ; 9(1): 132, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30644416

ABSTRACT

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.


Subject(s)
Hydroxybenzoates/pharmacology , Integrases/chemistry , Protein Aggregates/drug effects , Protein Domains/physiology , Spumavirus/enzymology , Viral Proteins/chemistry , Buffers , Integrases/metabolism , Osmolar Concentration , Protein Multimerization/drug effects , Viral Proteins/metabolism
6.
Anal Biochem ; 556: 78-84, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29932890

ABSTRACT

Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.


Subject(s)
Bacterial Proteins , Gene Expression , Hydroxybenzoates/chemistry , Optical Imaging , Protocatechuate-3,4-Dioxygenase , Pseudomonas putida , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Deoxyribonucleases , Enzyme Stability , Oxygen/chemistry , Protocatechuate-3,4-Dioxygenase/biosynthesis , Protocatechuate-3,4-Dioxygenase/chemistry , Protocatechuate-3,4-Dioxygenase/genetics , Protocatechuate-3,4-Dioxygenase/isolation & purification , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
7.
Nucleic Acids Res ; 45(2): 685-698, 2017 01 25.
Article in English | MEDLINE | ID: mdl-27738136

ABSTRACT

Wrapping of genomic DNA into nucleosomes poses thermodynamic and kinetic barriers to biological processes such as replication, transcription, repair and recombination. Previous biochemical studies have demonstrated that in the presence of adenosine triphosphate (ATP) the human RAD51 (HsRAD51) recombinase can form a nucleoprotein filament (NPF) on double-stranded DNA (dsDNA) that is capable of unwrapping the nucleosomal DNA from the histone octamer (HO). Here, we have used single molecule Förster Resonance Energy Transfer (smFRET) to examine the real time nucleosome dynamics in the presence of the HsRAD51 NPF. We show that oligomerization of HsRAD51 leads to stepwise, but stochastic unwrapping of the DNA from the HO in the presence of ATP. The highly reversible dynamics observed in single-molecule trajectories suggests an antagonistic mechanism between HsRAD51 binding and rewrapping of the DNA around the HO. These stochastic dynamics were independent of the nucleosomal DNA sequence or the asymmetry created by the presence of a linker DNA. We also observed sliding and rotational oscillations of the HO with respect to the nucleosomal DNA. These studies underline the dynamic nature of even tightly associated protein-DNA complexes such as nucleosomes.


Subject(s)
Histones/metabolism , Nucleosomes/metabolism , Rad51 Recombinase/metabolism , Adenosine Triphosphate/metabolism , DNA/genetics , DNA/metabolism , DNA Replication , Histones/chemistry , Humans , Hydrolysis , Models, Biological , Nucleoproteins/metabolism , Protein Binding , Protein Multimerization
8.
Nat Commun ; 6: 10209, 2015 Dec 18.
Article in English | MEDLINE | ID: mdl-26681117

ABSTRACT

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.


Subject(s)
Antibody Diversity/genetics , B-Lymphocytes/metabolism , Cytidine Deaminase/metabolism , DNA, Single-Stranded/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA/metabolism , Viral Proteins/metabolism , Animals , Antibody Diversity/immunology , B-Lymphocytes/immunology , Cytidine Deaminase/immunology , Escherichia coli , Fluorescence Resonance Energy Transfer , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Sf9 Cells , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/immunology , Spodoptera , Transcription, Genetic/genetics , Transcription, Genetic/immunology
10.
J Biol Chem ; 287(19): 15826-35, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22362763

ABSTRACT

APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C → U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3' → 5' direction. A similar polarity observed in vivo is believed responsible for initiating localized C → T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5' hot motif, a motif-dependent DNA contraction greatest for 5' hot > 3' hot > 5' cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.


Subject(s)
Cytidine Deaminase/metabolism , DNA, Single-Stranded/metabolism , Fluorescence Resonance Energy Transfer/methods , APOBEC-3G Deaminase , Bacteriophage M13/genetics , Base Sequence , Binding Sites/genetics , Biocatalysis , Cytidine Deaminase/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/metabolism , Deamination , Fluorescent Dyes/chemistry , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...