Research articles

Endonuclease V can initiate the repair of deaminated purine bases by recognizing them and hydrolyzing the second phosphodiester bond on their 3′ side. Now the crystal structures of endonuclease V in complex with its substrate and its product reveal a wedge motif acting as a minor groove–damage sensor and a pocket to recognize the lesion; the enzyme remains tightly bound to the 5′ phosphate product, perhaps to hand it over to downstream repair factors.

MIA40, found in the mitochondrial intermembrane space, is a central component in the import system that transports certain cysteine motif–containing proteins into the mitochondria. New analyses reveal that MIA40 forms a novel thioredoxin fold. Its redox center catalyzes the formation of the first disulfide bond of a substrate, causing the susbtrate's second disulfide to require only oxygen for its formation.

MicroRNA target sites tend to reside in the 3′ untranslated regions of transcripts in animals. By altering the stop codon position in a model target and thus placing miRNA target sites within open reading frames, it is now found that translation through a miRNA target region is refractory to miRNA-mediated repression.

Activation of the 20S proteasome requires the binding of regulatory proteins such as the 19S regulatory particle, which opens the 20S gates allowing substrate access to the active sites. New data now indicate that binding of a polyubiquitylated substrate to the 19S particle allows further opening of the 20S gates, suggesting a feedforward mechanism for 20S activation.

One of the key early steps in splicing is recognition of the 5′ splice site by base-pairing to the U1 small nuclear RNA. Data now indicate that U1 can shift to recognize what had been designated as atypical 5′ splice sites, broadening the scope of what can be recognized as a functional splice site by the canonical machinery and thus impacting both splicing predictions and mechanism, as well as providing a potential mechanism underlying a puzzling mutation associated with pontocerebellar hypoplasia.

RDE-1 is a Caenorhabditis elegans Argonaute homolog involved in mediating the primary response to small RNA interference. Analyses now indicate that the RNase H–homologous region of RDE-1 is not needed for target cleavage, but is specifically required for removing the passenger strand of fully complementary siRNA duplexes.

Fatty acid synthase is composed by several catalytic domains that work in sequence, with reaction intermediates being transferred between them. Single-particle EM analysis of different catalytic mutants of rat FAS imaged in the presence of substrates reveals the domains' movements during the reaction cycle.

Small RNAs are involved in the regulation of gene expression. During a hunt for microRNAs in the simple chordate Ciona intestinalis, a distinct class of small RNAs, offset from the microRNA expressed from a given hairpin, have been defined. These offset small RNAs are expressed in relative abundance in C. intestinalis and are also detected at specific developmental stages. Although their function is unclear, these results suggest that microRNA processing has distinct properties in this simple organism.

Nucleic acid polymerases catalyze nucleotidyl transfer reactions with two proton-transfer events, deprotonation of the 3′-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Functional analyses now show that the proton donor for the latter transfer is an active-site residue.

The Dsl1p tethering complex is crucial for Golgi-to-ER retrograde trafficking of vesicles in yeast. The crystal structures of two out of three Dsl1p complex components reveal similarity to exocyst and COG complex components, which act in tethering vesicles to the plasma membrane and Golgi, respectively, suggesting a conserved tethering strategy at several eukaryotic membranes.

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. These repeats adopt unusual structures that block DNA replication in vitro and in unicellular organisms. Mirkin and co-workers asked whether the same holds true in mammalian cells. They find that CGG repeats stall replication forks in a length-dependent, but orientation-independent, manner and do not seem to trigger an intra–S phase checkpoint response. They suggest that fragile sites arise because the cell cycle continues before replication is complete and the under-replicated areas would convert into constrictions and/or double-stranded breaks.

DNA packaging into nucleosomes presents a barrier to many motor proteins, including the transcriptional machinery. By unzipping DNA in single nucleosomes, a detailed map at near base pair resolution of histone-DNA interactions is now provided, suggesting that interaction with the two DNA strands is decoupled and that unraveling past the dyad axis of the nucleosome, as might occur when a motor protein passes through, is sufficient to displace histones.

Using CLIP-seq technology, the genome-wide binding sites of the FOX2 splicing regulator in human embryonic stem cells (hESCs) are now identified. Further work based on FOX2 depletion uncovers the underlying logic of FOX2-mediated regulation of alternative splicing and finds that such compromised hESCs undergo rapid cell death.

Endonuclease V can initiate the repair of deaminated purine bases by recognizing them and hydrolyzing the second phosphodiester bond on their 3′ side. Now the crystal structures of endonuclease V in complex with its substrate and its product reveal a wedge motif acting as a minor groove–damage sensor and a pocket to recognize the lesion; the enzyme remains tightly bound to the 5′ phosphate product, perhaps to hand it over to downstream repair factors.

Triple expansions underlie a number of human disorders. Triplet repeat instability in yeast mutants for the Srs2 and Sgs1 helicases indicate that recombination underlies instability in such a genetic background. Further analysis of replication intermediates indicates that Srs2 is likely to be involved in replication fork reversal within repetitive sequences, a process involved in preventing repeat instability and fragility.

Sortilin is a neuronal receptor involved in sorting and signal transduction. The crystal structure of the mature Sortilin ectodomain bound to one of its ligands, neurotensin, reveals a binding tunnel formed by the Sortilin β-propeller domain. Combined with binding and mutagenesis studies, the findings suggest that Sortilin substrates compete for access to the tunnel so that only one ligand binds at a time.

Myosins have roles in many biological processes that go beyond muscle contraction and vesicle transport, including furrowing during cytokinesis, signal transduction and RNA polymerase I–dependent transcription. Studying these various complex processes will require the use of isoform-specific small molecules that alter motor activity. The marine natural product pentabromopseudilin is now shown to act as an allosteric effector of myosin function and potent inhibitor of vertebrate myosin-5a–dependent motor activity.