The distribution pattern of proliferating cell nuclear antigen in the nuclei of Leishmania donovani

Abstract

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol d and Pol e. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

Leishmanicidal Activities of Novel Methylseleno-Imidocarbamates

“…Since both drugs (2b and 4b) significantly affected proliferation, we asked whether expression of genes related to proliferation would be altered. For this purpose, we selected PCNA, the gene for which is highly expressed in actively proliferating promastigotes (21). PCNA expression was then measured by quantitative RT-PCR in L. major promastigotes treated with 2.5 M 2b or 3 M 4b for 24 h. mRNA levels of PCNA were significantly downregulated in parasites exposed to 2.5 M 2b (P Ͻ 0.01; Fig.…”

“…Besides cell cycle control, it is involved in DNA replication, chromatin remodeling, and DNA repair (34). In the G 1 phase, PCNA was shown to be highly expressed in actively proliferating promastigotes throughout the cell cycle (21). During this phase, cells grow in size and prepare for division if the metabolic conditions are optimum.…”

A highly basic sequence at the N-terminal region is essential for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani

“…L. donovani genomic DNA was isolated by phenol/chloroform extraction followed by ethanol precipitation, as described previously (Kumar et al, 2009), and the ORC1 gene was cloned by amplification of the gene using Leishmania genomic DNA as template and Phu DNA polymerase (Finnzymes). Amplification was carried out using end primers designed based on the L. major genome sequence, ORC1-F and ORC1-R (Table 1).…”

Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

“…Expression and purification of rLdPCNA were previously reported for L. donovani strain S1 (29), but here entirely different conditions for expression and purification of rPCNA of the LdSR strain were optimized, and further characterization studies were conducted using this protein.…”