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, also interacting with each other directly in vitro. 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 here as well. 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 that associate with PCNA have been identified 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 may not be confined to trypanosomatids.

29DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase 30 whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in 31Leishmania donovani Cdc45. This motif is typically linked to interaction with the 32 eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can 33 potentially bind upto three different proteins simultaneously via a loop region present in 34 each monomer. Multiple binding partners have been identified from among the replication 35 machinery in other eukaryotes, and the concerted /sequential binding of these partners are 36 central to the fidelity of the replication process. Though conserved in Cdc45 across 37Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei 38Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such 39 an interaction in the in vivo context. Having confirmed the importance of Cdc45 in 40Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole 41 cell extracts, interacting with each other directly in vitro also. The interaction is mediated 42 via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance 43 of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to 44 be essential for cell survival in this organism also. Our results implicate a role for the 45 Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-46 PCNA interaction might help tether PCNA and associated replicative DNA polymerase to 47 Author Summary: 54Leishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The 55 prevalent form in the Indian subcontinent is visceral Leishmaniasis (VL), which is fatal if 56 not treated on time. While there are drugs for treatment, the hunt for additional drugs 57 continues due to emerging drug resistance patterns. The parasite is transmitted by the bite 58 of the sandfly, whereupon it establishes itself within cells of the host immune system 59 (macrophages) and reproduces by binary fission. The replication of its genome is essential 60 for parasite survival. Eukaryotic DNA replication is generally conserved across species. 61This study targets Cdc45, a protein that helps unwind the DNA double helix to enable 62 copying of the two strands into two daughter strands. The new chains of DNA are 63 synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen 64 (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to 65 interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our 66 results suggest this interaction is seen in some other eukaryotes as well. Based on the 67 results of our experiments we propose that Cdc45 may help moor PCNA-polymerase 68 complexes to template DNA. 69 70 71 72 73 74 75 76 77 78 HIV-Leishmania infections, and is deadly if not treated in a timely manner. In the Indian 83 subcontinent ...

Supporting informationS1 File. Supplementary methods. Contains descriptions of various methods used in the study. (DOCX)