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, 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.

Transcriptome Analysis of Caco-2 Cells upon the Exposure of Mycotoxin Deoxynivalenol and Its Acetylated Derivatives

“…PCNA is loaded at the primer template junction by the multi-subunit replication factor C (RFC) complex, which can open the PCNA ring and clamp it on the DNA. [ 50 , 51 , 52 , 53 ]. PCNA also works as an auxiliary protein of mammalian DNA polymerase δ (POLD) to stimulate the activity of the DNA polymerase δ core enzyme [ 54 ].…”

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6

“…Deletion or dysfunction of CDC45 leads to cell proliferation inhibition even cell death. [9][10][11] Here, we firstly confirmed knockout of cdc45 induced by CRISPR/Cas9 inhibits HEK293T cell proliferation. This gives evidence that CDC45 is important for cell cycle progression.…”

“… 5 , 6 , 7 , 8 Deletion of cdc45 induces decrease of cell cycle progression in Leishmania Donovani . 9 siRNA‐mediated knockdown of CDC45 could lead to cell proliferation inhibition and cell apoptosis. 10 , 11 Downregulation of cdc45 arrests cells in G1‐phase and represses the expression of G1‐/S‐phase related genes.…”

KAP1 phosphorylation promotes the survival of neural stem cells after ischemia/reperfusion by maintaining the stability of PCNA

“…PCNA is a core component of the eukaryotic replication complex with a unique ring tertiary structure. PCNA can bind to different replication-related proteins to coordinate the DNA replication process [26,[46][47][48]. PCNA also acts as a functional conversion factor, interacts with various cytokines through different regulatory modes, and participates in many important cellular events, including DNA damage repairment and refinement, cell cycle regulation, and apoptosis [46,[49][50][51].…”

“…PCNA can bind to different replication-related proteins to coordinate the DNA replication process [26,[46][47][48]. PCNA also acts as a functional conversion factor, interacts with various cytokines through different regulatory modes, and participates in many important cellular events, including DNA damage repairment and refinement, cell cycle regulation, and apoptosis [46,[49][50][51]. As an indicator of cell proliferation, PCNA correlates with the occurrence and development of diseases linked to cell proliferation [51][52][53].…”