Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation.
The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum. Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiaeorigin recognition complex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.
Ashish
A
Gupta
Parul
P
Mehra
Suman Kumar
SK
Dhar
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004261:DNA Replication; D015870:Gene Expression; D051738:Origin Recognition Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D052939:Schizonts / Q000378:metabolism
C497220:ORC5 protein, S cerevisiae; D051738:Origin Recognition Complex; D018809:Proliferating Cell Nuclear Antigen; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases