Trypanosome prereplication machinery contains a single functional orc1/cdc6 protein, which is typical of archaea.
In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.
Godoy
Patrícia Diogo de Melo
PD
Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo 05503900, Brazil.
Nogueira-Junior
Luis Antonio
LA
Paes
Lisvane S
LS
Cornejo
Alberto
A
Martins
Rafael Miyazawa
RM
Silber
Ariel M
AM
Schenkman
Sergio
S
Elias
M Carolina
MC
D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics* / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D051738:Origin Recognition Complex; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins