Origins of DNA replication.

In all kingdoms of life, DNA is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life

Structural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6

The origin recognition complex (ORC) is essential for loading the Mcm2–7 replicative helicase onto DNA during DNA replication initiation. Here, the authors describe several cryo-electron microscopy structures of Drosophila ORC bound to DNA and its cofactor Cdc6 and also report an in vitro reconstitution system for Drosophila Mcm2–7 loading, revealing unexpected features of ORC’s DNA binding and remodeling mechanism during Mcm2–7 loading

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis

Conformational control and DNA-binding mechanism of the metazoan origin recognition complex

In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2-7 (Mcm2-7) replicative helicase onto DNA to license origins. Drosophila ORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2-7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), we show that the autoinhibited state of Drosophila ORC is populated in solution, and that human ORC can also adopt this form. ATP binding to ORC supports a transition from the autoinhibited state to an active configuration, enabling the nucleotide-dependent association of ORC with both DNA and Cdc6. An unstructured N-terminal region adjacent to the conserved ATPase domain of Orc1 is shown to be required for high-affinity ORC-DNA interactions, but not for activation. ORC optimally binds DNA duplexes longer than the predicted footprint of the ORC ATPases associated with a variety of cellular activities (AAA+) and winged-helix (WH) folds; cryo-EM analysis of Drosophila ORC bound to DNA and Cdc6 indicates that ORC contacts DNA outside of its central core region, bending the DNA away from its central DNA-binding channel. Our findings indicate that ORC autoinhibition may be common to metazoans and that ORC-Cdc6 remodels origin DNA before Mcm2-7 recruitment and loading