Characterization of Leishmania donovani MCM4: expression patterns and interaction with PCNA

• Published in: PloS one 2011-07, Vol.6 (7), p.e23107
• Main Authors: Minocha, Neha, Kumar, Devanand, Rajanala, Kalpana, Saha, Swati
• Format: Article
• Language: English
• Subjects: Biology; Blotting, Western; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Nucleus - metabolism; Cell survival; Chromatin; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA damage; DNA replication; DNA, Protozoan - genetics; Drosophila; Elongation; Eukaryotes; Flow Cytometry; Fluorescent Antibody Technique; Immunoenzyme Techniques; Immunoprecipitation; Kinases; Leishmania; Leishmania donovani; Leishmania donovani - metabolism; Leishmaniasis; Localization; Mutagenesis, Site-Directed; Mutation; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Parasitic diseases; Phosphorylation; Proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - metabolism; Promastigotes; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Replication; Replication initiation; Replication Origin; S phase; S Phase - physiology; Saccharomyces cerevisiae; Viability; Xenopus; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0023107

Events leading to origin firing and fork elongation in eukaryotes involve several proteins which are mostly conserved across the various eukaryotic species. Nuclear DNA replication in trypanosomatids has thus far remained a largely uninvestigated area. While several eukaryotic replication protein orthologs have been annotated, many are missing, suggesting that novel replication mechanisms may apply in this group of organisms. Here, we characterize the expression of Leishmania donovani MCM4, and find that while it broadly resembles other eukaryotes, noteworthy differences exist. MCM4 is constitutively nuclear, signifying that, unlike what is seen in S.cerevisiae, varying subcellular localization of MCM4 is not a mode of replication regulation in Leishmania. Overexpression of MCM4 in Leishmania promastigotes causes progress through S phase faster than usual, implicating a role for MCM4 in the modulation of cell cycle progression. We find for the first time in eukaryotes, an interaction between any of the proteins of the MCM2-7 (MCM4) and PCNA. MCM4 colocalizes with PCNA in S phase cells, in keeping with the MCM2-7 complex being involved not only in replication initiation, but fork elongation as well. Analysis of a LdMCM4 mutant indicates that MCM4 interacts with PCNA via the PIP box motif of MCM4--perhaps as an integral component of the MCM2-7 complex, although we have no direct evidence that MCM4 harboring a PIP box mutation can still functionally associate with the other members of the MCM2-7 complex- and the PIP box motif is important for cell survival and viability. In Leishmania, MCM4 may possibly help in recruiting PCNA to chromatin, a role assigned to MCM10 in other eukaryotes.