title: Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases.
abstract: 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 Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) 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.
authors.affiliation: Structural Biology Laboratory, Sincrotrone Trieste, Trieste 34149, Italy.
authors.forename: Ivet
authors.initials: I
authors.lastname: Krastanova
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Sannino
authors.forename: Heinz
authors.initials: H
authors.lastname: Amenitsch
authors.forename: Opher
authors.initials: O
authors.lastname: Gileadi
authors.forename: Francesca M
authors.initials: FM
authors.lastname: Pisani
authors.forename: Silvia
authors.initials: S
authors.lastname: Onesti
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D008345:Manganese; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria; D010727:Phosphoric Diester Hydrolases