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

Abstract

The conserved eukaryotic DNA replication protein ORC1 is one of the constituents of prereplication complexes that assemble at or very near origins prior to replication initiation. ORC1 has been shown to be constitutively nuclear in Leishmania major. This study investigates the sequences involved in nuclear localization of ORC1 in Leishmania donovani, the causative agent of visceral leishmaniasis. Nuclear localization signals (NLSs) have been reported in only a few Leishmania proteins. Functional analyses have delineated NLSs to regions of~60 amino acids in length in the tyrosyl DNA phosphodiesterase I and type II DNA topoisomerase of L. donovani, and in the L. major kinesin KIN13-1. Using a panel of site-directed mutations we have identified a sequence essential for nuclear import of LdORC1. This sequence at the N terminus of the protein comprises residues 2-5 (KRSR), with K2, R3 and R5 being crucial. Independent mutation of the K2 residue causes exclusion of the protein from the nucleus, while mutating the R5 residue leads to diffusion of the protein throughout the cell. This sequence, however, is insufficient for targeting a heterologous protein (b-galactosidase) to the nucleus. Analysis of additional ORC1 mutations and reporter constructs reveals that while the highly basic tetra-amino acid sequence at the N terminus is essential for nuclear localization, the ORC1 NLS in its entirety is more complex, and of a distributive character. Our results suggest that nuclear localization signalling sequences in Leishmania nuclear proteins are more complex than what is typically seen in higher eukaryotes.

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

“…LdORC1/CDC6 has an N-terminus sequence essential for its nuclear import. This signaling sequence comprises residues 2-5 (KRSR), among which K2, R3, and R5 are crucial for LdORC1/CDC6 proper localization (Kumar et al, 2012). Importantly, ChIP-chip approaches revealed that ORC1/CDC6 co-localizes with fired replication origins in T. brucei (Tiengwe et al, 2012a), supporting the hypothesis that ORC1/CDC6 is the functional counterpart of the ORC complex.…”

“…Like in other protozoans, geminin homologous were not found in Trypanosomatidae (Table 1). Moreover, ORC1/CDC6 (Kumar et al, 2012), MCM2-7 (Dang & Li, 2011;Kumar et al, 2009;Minocha et al, 2011b), and GINS (Dang & Li, 2011) are expressed constitutively in the nucleus throughout the entire cell cycle, suggesting that the sub-localization of their respective complexes is not a regulative mechanism in these organisms. Interestingly, Li et al found that, in T. brucei, CDC45 is excluded from the nucleus after DNA replication (Dang & Li, 2011).…”

“…Interestingly, ORC1/CDC6 knockdown in T. brucei results in enucleated cells, suggesting defects in DNA replication (Godoy et al, 2009;Tiengwe et al, 2012b). An ORC1 ortholog has been identified in L. major and L. donovani (LdORC1/CDC6) (Kumar et al, 2008(Kumar et al, , 2012) and the protein is constitutively expressed in the nucleus, as described in trypanosomes. LdORC1/CDC6 has an N-terminus sequence essential for its nuclear import.…”

“…In yeast and metazoans, ORC is a six-subunit complex. Orthologues have been identified in many eukaryotes, such as Schizosaccharomyces pombe, Drosophila melanogaster, Xenopus laevis, and Homo sapiens (Bleichert et al, 2015;Kong et al, 2003;Kumar et al, 2012). ORC1-6 interacts with replication origins in an ATP-dependent manner (Bleichert et al, 2015;Zwerschke et al, 1994) and ORC-mediated ATP hydrolysis is essential for recruiting MCM proteins (Godoy et al, 2009).…”

Identification and Characterization of the Regions Involved in the Nuclear Translocation of the Heterodimeric Leishmanial DNA Topoisomerase IB

“…Only a few reports describe functional NLS in Leishmania [19–22]. Due to this, it is not possible to find a common sequence for nuclear transport in these parasites.…”