The catalytic triad of rice NARROW LEAF1 involves H234.

NARROW LEAF1 (NAL1) exerts a multifaceted influence on leaf morphology and crop yield. Recent crystal study proposed that histidine 233 (H233) is part of the catalytic triad. Here we report that unlike suggested previously, H234 instead of H233 is a component of the catalytic triad alongside residues D291 and S385 in NAL1.

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain.

The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions.

Slow motions in A·T rich DNA sequence.

In free B-DNA, slow (microsecond-to-millisecond) motions that involve equilibrium between Watson-Crick (WC) and Hoogsteen (HG) base-pairing expand the DNA dynamic repertoire that could mediate DNA-protein assemblies. R relaxation dispersion NMR methods are powerful tools to capture such slow conformational exchanges in solution using C/ N labelled DNA. Here, these approaches were applied to a dodecamer containing a TTAAA element that was assumed to facilitate nucleosome formation.

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States.

HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with nucleic acids, and p1 and p6, two unstructured regions, p6 containing the motifs to bind ALIX, the cellular ESCRT factor TSG101 and the viral protein Vpr. The processing of Gag by the viral protease subsequently liberates NCp15 (NC-p1-p6), NCp9 (NC-p1) and NCp7, NCp7 displaying the optimal chaperone activity of nucleic acids.

Structural Explorations of NCp7-Nucleic Acid Complexes Give Keys to Decipher the Binding Process.

A comprehensive view of all the structural aspects related to NCp7 is essential to understand how this protein, crucial in many steps of the HIV-1 cycle, binds and anneals nucleic acids (NAs), mainly thanks to two zinc fingers, ZF1 and ZF2. Here, we inspected the structural properties of the available experimental models of NCp7 bound to either DNA or RNA molecules, or free of ligand. Our analyses included the characterization of the relative positioning of ZF1 and ZF2, accessibility measurements and the exhaustive, quantitative mapping of the contacts between amino acids and nucleotides by a recent tessellation method, VLDM.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd.

H, C and N backbone and partial side-chain resonance assignments of the C-terminal domain of HIV-1 Pr55 encompassed in NCp15.

During HIV-1 assembly, the Pr55 polyprotein precursor (Gag) interacts with the genomic RNA, with lipids of the plasma membrane, with host proteins (ALIX, TSG101) through the ESCRT complex, with the viral protein Vpr and are involved in intermolecular interactions with other Pr55 proteins. This network of interactions is responsible for the formation of the viral particle, the selection of genomic RNA and the packaging of Vpr. The C-terminal domain of Gag encompassed in NCp15 is involved in the majority of these interactions, either by its nucleocapsid or its p6 domains.

Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex.

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out.

Identification of acylation products in SHAPE chemistry.

SHAPE chemistry (selective 2'-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2'-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC-MS/MS experiments.

Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding.

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method.

A 3'-5' exonuclease activity embedded in the helicase core domain of Candida albicans Pif1 helicase.

3'-5' exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3'-5' exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain.

Dynamics of Methylated Cytosine Flipping by UHRF1.

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase.

The intrinsic mechanics of B-DNA in solution characterized by NMR.

Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (ΔRDCs) involving C6/8-H6/8, C3'-H3' and C4'-H4' vectors are correlated to the(31)P chemical shifts (δP), which reflect the populations of the BI and BII backbone states.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing.

NMR studies of DNA support the role of pre-existing minor groove variations in nucleosome indirect readout.

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI ↔ BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5' half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities.

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1).

Retrospective on the all-in-one retroviral nucleocapsid protein.

This review aims at briefly presenting a retrospect on the retroviral nucleocapsid protein (NC), from an unspecific nucleic acid binding protein (NABP) to an all-in-one viral protein with multiple key functions in the early and late phases of the retrovirus replication cycle, notably reverse transcription of the genomic RNA and viral DNA integration into the host genome, and selection of the genomic RNA together with the initial steps of virus morphogenesis. In this context we will discuss the notion that NC protein has a flexible conformation and is thus a member of the growing family of intrinsically disordered proteins (IDPs) where disorder may account, at least in part, for its function as a nucleic acid (NA) chaperone and possibly as a protein chaperone vis-à-vis the viral DNA polymerase during reverse transcription. Lastly, we will briefly review the development of new anti-retroviral/AIDS compounds targeting HIV-1 NC because it represents an ideal target due to its multiple roles in the early and late phases of virus replication and its high degree of conservation.

Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription.

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer.

Mg2+ in the major groove modulates B-DNA structure and dynamics.

This study investigates the effect of Mg(2+) bound to the DNA major groove on DNA structure and dynamics. The analysis of a comprehensive dataset of B-DNA crystallographic structures shows that divalent cations are preferentially located in the DNA major groove where they interact with successive bases of (A/G)pG and the phosphate group of 5'-CpA or TpG. Based on this knowledge, molecular dynamics simulations were carried out on a DNA oligomer without or with Mg(2+) close to an ApG step.

Intrinsic nucleic acid dynamics modulates HIV-1 nucleocapsid protein binding to its targets.

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem.

Flexible nature and specific functions of the HIV-1 nucleocapsid protein.

One salient feature of reverse transcription in retroviruses, notably in the human immunodeficiency virus type 1, is that it requires the homologous nucleocapsid (NC) protein acting as a chaperoning partner of the genomic RNA template and the reverse transcriptase, from the initiation to the completion of viral DNA synthesis. This short review on the NC protein of human immunodeficiency virus type 1 aims at briefly presenting the flexible nature of NC protein, how it interacts with nucleic acids via its invariant zinc fingers and flanking basic residues, and the possible mechanisms that account for its multiple functions in the early steps of virus replication, notably in the obligatory strand transfer reactions during viral DNA synthesis by the reverse transcriptase enzyme.

Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein.

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA-DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer.