While a substantial body of literature bears testimony to the fact that DNA replication in eukaryotes is a highly conserved process, replication of the genomes of trypanosomatids has remained largely unexamined. We have endeavoured to investigate one of the replication proteins in Leishmania donovani
, the causative agent of the deadly disease visceral Leishmaniasis. In our study we find noteworthy differences with other eukaryotes. This is not surprising as several orthologs of eukaryotic replication proteins are missing in Leishmania
, as determined from annotation of the Leishmania
genome sequences 
. It is possible that novel Leishmania
proteins regulate the process in this organism.
Functional data has revealed that all six components of MCM2-7 contribute to the same activity. Immunodepletion experiments in Xenopus
extracts indicate that depleting any single MCM protein is sufficient to negatively impact DNA replication 
. We have cloned and characterized one of the six MCM2-7 proteins, MCM4. LdMCM4 has the conserved motifs associated with this protein (). The MCM2-7 belong to the family of AAA+ ATPases and LdMCM4 has the P-loop NTPase domain that characterizes members of this family (residues 437 to 589). While all the MCM2-7 belong to the AAA+ ATPase family and have ATP binding sites, individual MCM subunits do not display ATPase activity 
. Analysis of MCM4 expression in Leishmania
promastigote extracts revealed the presence of two bands near the expected size, and probing of MCM4-FLAG with phospho-antibodies suggests that the upper band corresponds to phosphorylated form (). MCM4 has been shown to be variably phosphorylated through the cell cycle in S. cerevisiae
, and mammalian cells 
, with chromatin-bound MCM4 undergoing specific phosphorylation in S phase. MCM4 phosphorylation in S phase in mammalian cells promotes the association of Cdc45 with chromatin as well 
. We found the phosphorylated form to be dominant throughout the cell cycle (), and it remains nuclear throughout the cell cycle (). However, site-specific phosphorylation events may be modulated in cell cycle dependent manner. Site-specific phosphorylations may also modulate MCM4 activity. Much more detailed investigations need to be carried out to address these issues.
The subcellular localization of MCM4 has been demonstrated to be a mode of replication regulation in S. cerevisiae 
, where nuclear export in S phase is believed to prevent re-replication from occurring. We found Leishmania
to be different in that MCM4 remained in the nucleus throughout the cell cycle (, ). In this, it resembles mammalian cells, where cell cycle progression does not affect MCM4 localization. Interestingly, the overexpression of MCM4 in Leishmania
resulted in a shortened nuclear S phase, with cells reaching G2/M faster than usual (). This suggests the possibility that cellular MCM4 levels may be limiting, an attribute of the protein that may play a role in modulating cell cycle progression.
Investigating possible interactions between MCM4 and PCNA revealed that while purified recombinant MCM4 expressed in E.coli
by itself does not bind to PCNA (data not shown), MCM4 in whole cell lysates interacts with PCNA (albeit somewhat weakly) ( and ). PCNA interacts only with the phosphorylated form of MCM4 (), indicating this as the likely reason why we were unable to detect the interaction in direct pulldowns between the two recombinant proteins expressed in E.coli
. It is also possible that MCM4 needs to be part of the MCM2-7 complex for the interaction to occur, although we have no experimental evidence of this. The MCM4-PCNA interaction appears to be ATP-independent, and while it is possible that the MCM4-PCNA interaction is mediated through other protein(s), we find that mutating a sequence in MCM4 that has been shown to be directly responsible for protein-PCNA interactions in other proteins (PIP box), results in the loss of the MCM4-PCNA interaction. As no other role has been assigned to this motif to date, this data suggests that the interaction is between MCM4 and PCNA (perhaps as part of the MCM2-7 holocomplex, though there is no direct evidence that the MCM4-PIP box mutant can still form a functional complex with the other members of the MCM2-7 proteins). The PIP domain is important for cell viability, as overexpression of MCM4-PIP box mutant that cannot interact with PCNA () results in overall decreased viability of Leishmania
cultures (). One interesting feature we observed was that the MCM4/PIP-FLAG protein seems to have a partial phosphorylation defect. As one of the residues mutated in the PIP box is a tyrosine residue, and tyrosine phosphorylation is detected in MCM4 (), it is possible that this residue is a site of phosphorylation in the protein. However, much more needs to be done to ascertain this. While PCNA in other eukaryotes has been shown to interact with several proteins involved in DNA repair and replication 
, to date no interactions with any of the MCM2-7 have been detected in any eukaryote. In S. cerevisiae
, MCM10 has been shown to interact with PCNA 
. This interaction is through the MCM10 PIP box, and an MCM10 PIP box mutant displays a severely defective cell growth and proliferation phenotype. MCM10 also interacts with MCM2-7 
, ORC 
and DNA polymerase α/primase complexes 
, and promotes initiation of replication as well as elongation. The loading of PCNA onto chromatin is believed to be facilitated via its interaction with MCM10, which is loaded onto pre-RCs after MCM2-7 
. In Leishmania,
the role of facilitation of chromatin loading of PCNA may be played by the MCM2-7 complex itself. Alternatively, the MCM2-7 complex and PCNA may be associated during elongation, as both proteins move along with the replication fork, MCM2-7 being the replicative helicase, and PCNA being the DNA polymerase δ processivity factor. Studies carried out by several research groups have shown that the MCM2-7 do not colocalize with PCNA in other eukaryotes 
. However, during Drosophila
chorion gene amplification, MCM2-7 immunocolocalized with PCNA at all stages of amplification 
indicating that these MCMs are present at chorion amplicons through replication initiation as well as elongation of replication forks. The observation that MCM4-GFP in Leishmania
promastigotes immunocolocalizes with PCNA in cells that are in S phase (), is suggestive of the possibility that the MCM2-7 move along with the elongating replication forks in Leishmania
. A role for MCM2-7 in replication fork elongation has been demonstrated in higher eukaryotes. Chromatin immunoprecipitation experiments demonstrate that the MCMs are displaced from origins and, along with Cdc45, move ahead of the replication fork, in contrast to the ORC proteins which do not 
. Experiments where the MCMs were destroyed after replication initiation indicated the continued necessity of MCMs in DNA replication even post-initiation 
. The colocalization of MCM4 and PCNA in Leishmania
is an aspect that needs to be further investigated by examining the behavior of endogenous MCM4, to rule out the possibility of our observations being the result of overexpression.
The data presented here describe the findings of an investigation of one of the key players of eukaryotic DNA replication in the protozoan Leishmania donovani, and lay the foundation for future studies directed at addressing the role of the MCM4-PCNA interaction in DNA replication. The results of our study underline the importance of studying DNA replication in non-conventional organisms as much as in model systems, and uncover facets of emerging diversities in modes of replication among eukaryotes.