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SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication. [ABSTRACT FROM AUTHOR]

Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment. [ABSTRACT FROM AUTHOR]

Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase. [ABSTRACT FROM AUTHOR]

Background: Splicing and alternate splicing are the two key biological processes that result in the generation of diverse transcript and protein isoforms in Plasmodium falciparum as well as in other eukaryotic organisms. Not much is known about the organization of splicing machinery and mechanisms in human malaria parasite. Present study reports the organization and assembly of Plasmodium spliceosome Sm core complex. Methods: Presence of all the seven Plasmodium Sm-like proteins in the intra-erythrocytic stages was assessed based on the protein(s) expression analysis using immuno-localization and western blotting. Localization/co-localization studies were performed by immunofluorescence analysis on thin parasite smear using laser scanning confocal microscope. Interaction studies were carried out using yeast two-hybrid analysis and validated by in vitro pull-down assays. PfPRMT5 (arginine methyl transferase) and PfSmD1 interaction analysis was performed by pull-down assays and the interacting proteins were identified by MALDI-TOF spectrometry. Results: PfSm proteins are expressed at asexual blood stages of the parasite and show nucleo-cytoplasmic localization. Protein-protein interaction studies showed that PfSm proteins form a heptameric complex, typical of spliceosome core complex as shown in humans. Interaction of PfSMN (survival of motor neuron, tudor domain containing protein) or PfTu-TSN (Tudor domain of Tudor Staphylococcal nuclease) with PfSmD1 proteins was found to be methylation dependent. Co-localization by immunofluorescence and co-immunoprecipitation studies suggested an association between PfPRMT5 and PfSmD1, indicating the role of arginine methylation in assembly of Plasmodium spliceosome complex. Conclusions: Plasmodium Sm-like proteins form a heptameric ring-like structure, although the arrangement of PfSm proteins slightly differs from human splicing machinery. The data shows the interaction of PfSMN with PfSmD1 and this interaction is found to be methylation dependent. PfPRMT5 probably exists as a part of methylosome complex that may function in the cytoplasmic assembly of Sm proteins at asexual blood stages of P. falciparum. [ABSTRACT FROM AUTHOR]

Abstract: Tudor Staphylococcal Nuclease (p100 or SND1), a member of the micronuclease family is a multifunctional protein that plays a key role(s) in transcription and splicing processes in many eukaryotic cells. PfTudor-SN, a Plasmodium homolog of the human p100 protein is a structurally conserved protein; however molecular details of its function are not yet understood. Our previous studies have shown that PfTudor-SN binds RNA and it is possible to selectively inhibit parasite growth by PfTudor-SN specific drugs. In the present study, we identified the molecular interactions between Plasmodium falciparum Tudor-SN and twelve Plasmodium proteins such as Histone h2A, SPT2 (a transcriptional regulator), a Cold-shock DNA binding protein in a bacterial two-hybrid screen. To get further insight into some of these interactions, we mapped the interaction domain in PfTudor-SN protein using the yeast two-hybrid system. Of these proteins, Plasmodium N-methyl-d-aspartate receptor associated protein, PfUbiquitin conjugating enzyme and Cold-shock DNA binding protein showed interaction with the SN domains of PfTudor-SN. Immuno-localization studies of the interacting proteins showed their presence predominantly in the nucleus, which inevitably suggests the molecular interactions between these proteins and PfTudor-SN. Furthermore, we also identified a molecular interaction between the Tudor domain of PfTudor-SN protein and Plasmodium spliceosomal Sm protein, PfSmD1 advocating the role of PfTudor-SN in the spliceosome assembly. Together, these results suggest multiple role(s) for PfTudor-SN protein mainly in nuclear and splicing processes at asexual blood stages of the malaria parasite. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium falciparum Tudor Staphylococcal Nuclease (PfTSN) has a multidomain organization and preferentially cleaves single stranded RNAs. PfTSN is quite distinct from its vertebrate homologues both in terms of its primary sequence and functional activity. Here, we analyzed the effect of PfTSN specific siRNA on parasite growth and development. Treatment of parasite culture with PfTSN siRNA at the late ring stage resulted in substantial inhibition in parasite growth. The PfTSN siRNA treated parasite cultures showed significant reduction in specific mRNA and PfTSN expression. Morphological examination of PfTSN siRNA treated parasites showed block in the development of parasite at the trophozoite stage. Treatment of parasites with a specific inhibitor of micrococcal nucleases, 3′,5′-deoxythymidine biphosphate (pdTp) resulted in similar block in parasite development, thereby suggesting that PfTSN plays an important role at the trophozoite stage of the parasite. Collectively, our findings point towards an essential role for the PfTSN in the parasite’s infection cycle. [Copyright &y& Elsevier]

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects. [ABSTRACT FROM AUTHOR]