Abstract
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.
Highlights
Eukaryotic DNA replication involves the licensing and activation of multiple origins
The MCM4 gene was amplified from Leishmania donovani genomic DNA using primers that were designed based on the Leishmania major genome sequence [16] as sequence information from Leishmania donovani was not available at the time
Like all the members of the MCM family, LdMCM4 carries the,200 amino acid MCM box (Figure 1A), within this domain there is a short region of sequence insertion (13 amino acids in length) in comparison with other eukaryotes
Summary
Eukaryotic DNA replication involves the licensing and activation of multiple origins. Origins are licensed by the assembly of pre-replication complexes (pre-RCs) in G1 phase [1,2,3], a process involving the ordered loading of ORCs 1-6, Cdc, Cdt, MCM27, and MCM10. GINS, Sld, Dpb and Cdc associate with the complexes to trigger origin activation, and with the recruitment of the elongation machinery, DNA synthesis commences [4,5]. The trypanosomatid Leishmania causes the group of diseases collectively called Leishmaniasis. Leishmaniasis occurs in three main forms – cutaneous, subcutaneous and visceral, and different Leishmania species cause different forms of the disease. Leishmania donovani is one of the causative agents of VL, prevalent in Sudan and the Indian subcontinent
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