Diamond nanocrystals emit bright fluorescence at 600-800 nm after irradiation by a 3 MeV proton beam (5 x 1015 ions/cm2) and annealing at 800 degrees C (2 h) in vacuum. The irradiation/annealing process yields high concentrations of nitrogen-vacancy defect centers ( approximately 107 centers/mum3), making possible visualization of the individual 100 nm diamond crystallites using a fluorescence microscope. The fluorescent nanodiamonds (FND) show no sign of photobleaching and can be taken up by mammalian cells with minimal cytotoxicity. The nanomaterial can have far-reaching biological applications.

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

SummaryInternal modification of RNAs with N6-methyladenosine (m6A) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts in a single-stranded context, the substrate requirements and physiological roles of the second m6A writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking Mettl16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in ∼64-cell blastocysts that are unfit for further development. This highlights the role of an m6A RNA methyltransferase in facilitating early development via regulation of SAM availability.

SummaryHuman APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of HIV-1, other retroviruses and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1 helix (residues 201-206) that was not apparent in the shorter protein and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5 and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different than the continuous β2 strand of another family member APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain including the inter-domain linker and some of the last α-helix. These structured residues (191)(192)(193)(194)(195)(196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N-and C-terminal β2 regions are too distant from one another to participate in this interaction.

No abstract

Two-photon fluorescence spectroscopy of negatively charged nitrogen-vacancy [(N-V)-] centers in type Ib diamond single crystals have been studied with a picosecond (7.5 ps) mode-locked Nd:YVO(4) laser operating at 1064 nm. The (N-V)- centers were produced by radiation damage of diamond using a 3 MeV proton beam, followed by thermal annealing at 800 degrees C. Prior to the irradiation treatment, infrared spectroscopy of the C-N vibrational modes at 1344 cm(-1) suggested a nitrogen content of 109 +/- 10 ppm. Irradiation and annealing of the specimen led to the emergence of a new absorption band peaking at approximately 560 nm. From a measurement of the integrated absorption intensity of the sharp zero-phonon line (637 nm) at liquid nitrogen temperature, we determined a (N-V)- density of (4.5 +/- 1.1) x 10(18) centers/cm3 (or 25 +/- 6 ppm) for the substrate irradiated at a dose of 1 x 1016) H(+)/cm(2). Such a high defect density allowed us to observe two-photon excited fluorescence and measure the corresponding fluorescence decay time. No significant difference in the spectral feature and fluorescence lifetime was observed between one-photon and two-photon excitations. Assuming that the fluorescence quantum yields are the same for both processes, a two-photon absorption cross section of sigma(TPA) = (0.45 +/- 0.23) x 10(-50) cm(4).s/photon at 1064 nm was determined for the (N-V)- center based on its one-photon absorption cross section of sigma(OPA) = (3.1 +/- 0.8) x 10(-17) cm2 at 532 nm. The material is highly photostable and shows no sign of photobleaching even under continuous two-photon excitation at a peak power density of 3 GW/cm(2) for 5 min.

No abstract

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd's deamination reactions within a 5=-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5=-CCC substrate over 5=-CCU substrate, which explains why A3G displays almost no processivity within a 5=-CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5=-CCC flanked on both sides by a single nucleotide. A3Gctd as well as fulllength A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo. Human APOBEC3G (A3G) is a member of a family of Zn 2ϩ -dependent polynucleotide cytosine deaminases. This family was named after APOBEC1 (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1) and also includes the antibody gene diversification enzyme AID (activation-induced cytidine deaminase) (reviewed in references 1-5). A3G can restrict HIV-1 replication by packaging into assembling viral particles for delivery to target cells, where it deaminates cytosine to uracil in newly transcribed viral DNA. These cDNA uracils base pair with adenine during plus-strand synthesis and result in G-to-A hypermutation and, in turn, inactivation of the viral genome. A3G has two Zn 2ϩ binding domains that span residues 1 to 196 and 197 to 384, but only the C-terminal domain is catalytically active (6-8). The Nterminal domain interacts with HIV-1 Vif, RNA, and singlestranded DNA (ssDNA) (e.g., see references 7 and 9-11). A3G predominantly deaminates the 3= cytosine (underlined) in a 5=-CCC sequence, although the middle cytosine can also be deaminated in subsequent reactions following deamination of the 3= cytosine (12-16). In longer ssDNA substrates with multiple 5=-CCC sites, A3G deamination exhibits a 3=¡5= spatial preference in vitro (9,17,18). In the present study, we use the catalytic domain of A3G (A3Gctd) to determine kinetic parameters. Our results provide kinetic constants for two independent deaminations within a 5=-CCC sequence, which explain A3G's catalytic site preference for the 3= cytosine. We identify a strong pH dependence of the reaction speed, which implies that a histidine residue is involved in substrate binding. In addition, we identify the shortestlength ssDNA substrate for A3Gctd to be a pentanucleotide. MATERIALS AND METHODSPurification of A3Gctd. The APOBEC3G C-terminal domain (A3Gctd), comprising amino acids 191 to 384, was expressed and purified as previously described (19). Briefly, the glutathione S-transferase (GST)-fused A3Gctd was...