Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication, as well as the progression of the replication fork. With GINS, Cdc45 is an essential co-factor 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. Here 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 DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyse the hydrolysis of phosphodiester bonds via a mechanism involving two Mn2+ ions. Only a subset of the amino acids that coordinate Mn2+ are 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.
Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases
Sannino V;Pisani F M;
2012
Abstract
Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication, as well as the progression of the replication fork. With GINS, Cdc45 is an essential co-factor 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. Here 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 DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyse the hydrolysis of phosphodiester bonds via a mechanism involving two Mn2+ ions. Only a subset of the amino acids that coordinate Mn2+ are 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
