DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival

Abstract

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, interacting with each other directly in vitro also. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival in this organism also. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins have been identified to associate with PCNA in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it is not confined to trypanosomatids.\n\nAuthor SummaryLeishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The prevalent form in the Indian subcontinent is visceral Leishmaniasis (VL), which is fatal if not treated on time. While there are drugs for treatment, the hunt for additional drugs continues due to emerging drug resistance patterns. The parasite is transmitted by the bite of the sandfly, whereupon it establishes itself within cells of the host immune system (macrophages) and reproduces by binary fission. The replication of its genome is essential for parasite survival. Eukaryotic DNA replication is generally conserved across species. This study targets Cdc45, a protein that helps unwind the DNA double helix to enable copying of the two strands into two daughter strands. The new chains of DNA are synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our results suggest this interaction is seen in some other eukaryotes as well. Based on the results of our experiments we propose that Cdc45 may help moor PCNA-polymerase complexes to template DNA.