Silvia Onesti

Hexameric ring structure of the full‐length archaeal MCM protein complex

In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three‐dimensional reconstruction of the full‐length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring‐shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double‐stranded DNA. The crystal structure of the amino‐terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy‐terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double‐stranded DNA translocases.

The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli

BACKGROUND Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. RESULTS The crystal structure of E. coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. CONCLUSIONS A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine.

The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well‐characterized C‐terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S‐adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV‐visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site‐directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.

Structure of an Archaeal Homolog of the Eukaryotic RNA Polymerase II RPB4/RPB7 Complex

The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated two-domain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript.

Structural biology of MCM helicases

The eukaryotic MCM2-7 complex is recruited onto origins of replication during the G1 phase of the cell cycle and acts as the main helicase at the replication fork during the S phase. Over the last few years a number of structural reports on MCM proteins using both electron microscopy and protein crystallography have been published. The crystal structures of two (almost) full-length archaeal homologs provide the first atomic pictures of a MCM helicase. However one of the structures is at low resolution and the other is of an inactive MCM. Moreover, both proteins are monomeric in the crystal, whereas the activity of the complex is critically dependent on oligomerization. Lower resolution structures derived from electron microscopy studies are therefore crucial to complement the crystallographic analysis and to assemble the multimeric complex that is active in the cell. A critical analysis of all the structural results elucidates the potential conformational changes and dynamic behavior of MCM helicase to provide a first insight into the gamut of molecular configurations adopted during the processes of DNA melting and unwinding.

Insights into the Architecture of the Replicative Helicase from the Structure of an Archaeal MCM Homolog

The minichromosome maintenance (MCM) proteins, members of the AAA+ (ATPase associated with diverse cellular activities) superfamily, are believed to constitute the replicative helicase in eukaryotic and archaeal species. Here, we present the 1.9 A resolution crystal structure of a monomeric MCM homolog from Methanopyrus kandleri, the first crystallographic structure of a full-length MCM. We also present an 18 A cryo-electron microscopy reconstruction of the hexameric MCM from Methanothermobacter thermautotrophicus, and fit the atomic resolution crystal structure into the reconstruction in order to generate an atomic model for the oligomeric assembly. These structural data reveal a distinct active site topology consisting of a unique arrangement of critical determinants. The structures also provide a molecular framework for understanding the functional contributions of trans-acting elements that facilitate intersubunit crosstalk in response to DNA binding and ATP hydrolysis.

Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity

The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP·AlFx treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.

Crystal Structure and RNA Binding of the Rpb4/Rpb7 Subunits of Human RNA Polymerase II.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have obtained crystals of the human Rpb4/Rpb7 heterodimer and determined the structure to 2.7 Å resolution. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the heterodimer close to the RNA exit groove in the 12 subunit yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies directed at dissecting the RNA-binding properties of the human Rpb4/Rpb7 complex and that of the homologous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent-exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4–B5 loop, but also extends towards the N-terminal domain.

The MCM complex: (just) a replicative helicase?

The MCM2-MCM7 (minichromosome maintenance 2-7) complex is involved both in the initiation and the elongation step of eukaryotic DNA replication and is believed to be the replicative helicase. Whereas the mechanism of DNA unwinding at the replication fork has been extensively investigated, the role of the MCM2-MCM7 complex during initiation has not yet been characterized by biochemical studies. Here we summarize the in vivo evidence which supports a role for the MCM complex in origin melting. In addition, we present an overview of the mechanism of action of a number of AAA+ (ATPase associated with various cellular activities) initiators and hexameric helicases, which can be used in turn as models for the steps of recognition, duplex melting, loading and nucleic acid translocation of the MCM helicase.

Structural and Functional Insights into the DNA Replication Factor Cdc45 Reveal an Evolutionary Relationship to the DHH Family of Phosphoesterases

Background: Although Cdc45 is a key replication factor, there are no biochemical or structural studies on the isolated protein. Results: We report the first purification and biochemical characterization of human Cdc45, as well as the first structural data on the isolated Cdc45 by small angle x-ray scattering. Conclusion: Cdc45 is related to the RecJ/DHH family of phosphoesterases and binds single-stranded DNA. Significance: The similarity has important evolutionary implications. Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2–7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn2+ ions. Only a subset of the amino acids that coordinates Mn2+ is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.