title: Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture.
abstract: DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.
authors.affiliation: The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Calvin
authors.initials: C
authors.lastname: Tiengwe
authors.forename: Lucio
authors.initials: L
authors.lastname: Marcello
authors.forename: Helen
authors.initials: H
authors.lastname: Farr
authors.forename: Catarina
authors.initials: C
authors.lastname: Gadelha
authors.forename: Richard
authors.initials: R
authors.lastname: Burchmore
authors.forename: J David
authors.initials: JD
authors.lastname: Barry
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Bell
authors.forename: Richard
authors.initials: R
authors.lastname: McCulloch
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000235:genetics; D034622:RNA Interference; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D051738:Origin Recognition Complex; D021122:Protein Subunits; D004265:DNA Helicases