Error thresholds for self- and cross-specific enzymatic replication

The information content of a non-enzymatic self-replicator is limited by Eigen's error threshold. Presumably, enzymatic replication can maintain higher complexity, but in a competitive environment such a replicator is faced with two problems related to its twofold role as enzyme and substrate: as enzyme, it should replicate itself rather than wastefully copy non-functional substrates, and as substrate it should preferably be replicated by superior enzymes instead of less-efficient mutants. Because specific recognition can enforce these propensities, we thoroughly analyze an idealized quasispecies model for enzymatic replication, with replication rates that are either a decreasing (self-specific) or increasing (cross-specific) function of the Hamming distance between the recognition or "tag" sequences of enzyme and substrate. We find that very weak self-specificity suffices to localize a population about a master sequence and thus to preserve its information, while simultaneous localization about complementary sequences in the cross-specific case is more challenging. A surprising result is that stronger specificity constraints allow longer recognition sequences, because the populations are better localized. Extrapolating from experimental data, we obtain rough quantitative estimates for the maximal length of the recognition or tag sequence that can be used to reliably discriminate appropriate and infeasible enzymes and substrates, respectively.Comment: 23 pages, 7 figures; final version as publishe

Structural basis for DNMT3A-mediated de novo DNA methylation.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease

Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase

This research was supported by the German Academic Exchange Service (DAAD), the Wellcome Trust (TKS and WNH as Trust Senior Research fellows), the Biotechnology and Biological Science Research Council (Structural Proteomics of Rational Targets) and the European Synchrotron Radiation Facility.Background: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)-over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of ( R)mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha 2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.Publisher PDFPeer reviewe

The use of self-assembled receptor layers in immunosensors

We demonstrate that a self-assembled monolayer consisting of an alkylthiol coupled synthetic peptide adsorbed to a gold substrate results in a sensitive receptor surface for specific recognition of protein molecules. The affinity constant of this binding is comparable with that of an antibody-antigen reaction. Evidence is found that such a receptor surface is capable of reversible binding

A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts

Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition

The structure-specific endonuclease activity of the human XPF–ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF–ERCC1 cleaves DNA structures such as stem–loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem–loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF–ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3′-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix–hairpin–helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF–HLD shows a preference for binding stem–loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF–ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity

The Exosome Subunit Rrp44 Plays a Direct Role in RNA Substrate Recognition

The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNAiMet lacking a single m1A58 modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNAiMet, showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNAiMet by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein

Chemical reactivity imprint lithography on graphene: Controlling the substrate influence on electron transfer reactions

The chemical functionalization of graphene enables control over electronic properties and sensor recognition sites. However, its study is confounded by an unusually strong influence of the underlying substrate. In this paper, we show a stark difference in the rate of electron transfer chemistry with aryl diazonium salts on monolayer graphene supported on a broad range of substrates. Reactions proceed rapidly when graphene is on SiO_2 and Al_2O_3 (sapphire), but negligibly on alkyl-terminated and hexagonal boron nitride (hBN) surfaces. The effect is contrary to expectations based on doping levels and can instead be described using a reactivity model accounting for substrate-induced electron-hole puddles in graphene. Raman spectroscopic mapping is used to characterize the effect of the substrates on graphene. Reactivity imprint lithography (RIL) is demonstrated as a technique for spatially patterning chemical groups on graphene by patterning the underlying substrate, and is applied to the covalent tethering of proteins on graphene.Comment: 25 pages, 6 figure