ORC Proteins Research Articles

Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

G9a Responds to KRAS‐Mediated Replication Stress by Increased Origin Licensing

Pancreatic Ductal Adenocarcinoma (PDAC) remains as one of the leading causes of cancer death in the US. KRAS mutations are recognized as the leading oncogenic driver of PDAC growth. Activation of KRAS causes increased proliferation that requires a level of adaptation during processes, such as DNA replication. This rapid duplication of the genome and epigenome generates havoc in the timing and coordination of replication origin firing, triggering a response known as replication stress (RS). The epigenomic regulator G9a is responsible for catalyzing histone H3 lysine 9 mono‐ and di‐methylation (H3K9me1 and H3K9me2). G9a is abundantly present in many forms of cancer, including PDAC, and because of its interaction with replication proteins, such as RPA and PCNA, it is known to have an important role during DNA replication. DNA replication happens at the S phase of the cell cycle, which requires that replication origins are recognized at late G1 phase by the origin recognition complex (ORC) followed by assembly of the pre‐replication complex (pre‐RC), comprised of CDC6, cdt1 and mini chromosome maintenance (MCM) proteins, to allow origin licensing and subsequent firing. We have shown that the G9a complex increases in response to KRAS activation and is critical to the growth‐promoting effects of this oncogene in PDAC. Though it is known that KRAS reduces distances between replication origins, there has been a paucity of knowledge regarding the role of G9a during replication origin licensing and whether it is directly involved with the pre‐RC. In the current study, we aim to better understand whether G9a interacts with the replication origins and the pre‐RC to increase origin licensing under conditions of KRAS‐mediated RS. To simulate the early events in oncogene‐induced RS to study compensatory mechanisms, we generated an inducible KRAS model (iKRAS‐HPNE) in an hTERT‐immortalized human non‐cancerous pancreatic ductal cell line, hTERT‐HPNE E6/E7. We found that KRAS induction for 48h in this iKRAS‐HPNE model activated the ATR RS‐response pathway, indicated by increased levels of P‐T1989‐ATR, P‐S345‐CHK1 and P‐S33‐RPA32 by western blot and immunofluorescence‐based microscopy. Concurrently, members of the G9a epigenomic complex (G9a, GLP and WIZ) significantly increased along with two subunits of the ORC (ORC1 and ORC2) and several other pre‐RC proteins (Cdt1, MCM2, MCM5 and MCM7). Co‐immunoprecipitation of G9a in the iKRAS‐HPNE cells revealed that G9a interacts with ORC2 protein under conditions of oncogene‐induced RS. Pharmacological inhibition of G9a with UNC0642 during KRAS‐induced RS resulted in a decrease of chromatin‐bound ORC1 and ORC2 as well as pre‐RC proteins in subcellular fractionation experiments, suggesting that functional G9a is critical for enhanced origin licensing. In summary, our results demonstrate that G9a directly interacts with replication origins and the pre‐RC during origin licensing under conditions of RS. Our study reveals one potential mechanism by which cancerous cells with KRAS mutation meet the demands for rapid cellular proliferation during RS and reinforces the role of G9a as a promising therapeutic target for PDAC.

RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle.

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

Identification and in silico analysis of the origin recognition complex in the human fungal pathogen Candida albicans.

DNA replication in eukaryotes is initiated by the orchestrated assembly and association of initiator proteins (heterohexameric Origin Recognition Complex, ORC) on the replication origins. These functionally conserved proteins play significant roles in diverse cellular processes besides their central role in ignition of DNA replication at origins. Candida albicans, a major human fungal pathogen, is a diploid budding yeast that belongs to Ascomycota. However, C. albicans is significantly diverged from a well-studied model organism Saccharomyces cerevisiae, another ascomycete. The components of the DNA replication machinery in C. albicans remain largely uncharacterized. Identification of factors required for DNA replication is essential for understanding the evolution of the DNA replication machinery. We identified the putative ORC homologs in C. albicans and determined their relatedness with those of other eukaryotes including several yeast species. Our extensive in silico studies demonstrate that the domain architecture of CaORC proteins share similarities with the ORC proteins of S. cerevisiae. We dissect the domain organization of ORC (trans-acting factors) subunits that seem to associate with DNA replication origins in C. albicans. We present a model of the 3D structure of CaORC4 to gain further insights of this protein’s function.

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1-6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2-7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9-mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2-7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2-7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2-7 to origins independent of ORC.

Enhanced transcription activity of histone acetyltransferase HBO1 after genotoxic stress

Chromatin remodeling is induced at sites of DNA damage as part of the damage response to allow DNA repair protein accumulation and stability of function. Histone posttranslational modifications are required to orchestrate DNA damage response, and ubiquitination of histone H2A-K15 at DNA double-strand break sites is involved in the recruitment of DNA repair protein 53BP1 to DNA damage sites. Acetylation of histone H4K16 promotes homologous recombination repair by inducing dissociation of 53BP1, while deacetylation inhibits the homologous recombination repair by promoting accumulation of 53BP1 at DNA damage sites. Chromatin remodeling is indispensable for accumulation of numerous DNA damage repair proteins and their subsequent dissociation, and we investigated the possibility of new DNA damage response pathway. In this study, we used qPCR, western blotting and survival assays to demonstrate that genotoxic stress, both biochemical and radiological, specifically upregulated the expression of histone acetyltransferase binding to ORC (HBO1) protein in the histone acetyltransferase MYST family, which colocalized with the DNA damage response protein, Fanconi anemia group D2 (FANCD2). HBO1 expression was upregulated via phosphoinositide-3-kinase-related kinase protein, which plays a crucial role as a DNA damage response signal. These results suggest that HBO1induced by genotoxic stress has a critical role in DNA damage responses.

Molecular and genetic organization of bands and interbands in the dot chromosome of Drosophila melanogaster

The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5′UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P–elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication.

DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein) leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.

Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex.

Ndt80 activates the meiotic ORC1 transcript isoform and SMA2 via a bi-directional middle sporulation element in Saccharomyces cerevisiae

ABSTRACTThe origin of replication complex subunit ORC1 is important for DNA replication. The gene is known to encode a meiotic transcript isoform (mORC1) with an extended 5′-untranslated region (5′-UTR), which was predicted to inhibit protein translation. However, the regulatory mechanism that controls the mORC1 transcript isoform is unknown and no molecular biological evidence for a role of mORC1 in negatively regulating Orc1 protein during gametogenesis is available. By interpreting RNA profiling data obtained with growing and sporulating diploid cells, mitotic haploid cells, and a starving diploid control strain, we determined that mORC1 is a middle meiotic transcript isoform. Regulatory motif predictions and genetic experiments reveal that the activator Ndt80 and its middle sporulation element (MSE) target motif are required for the full induction of mORC1 and the divergently transcribed meiotic SMA2 locus. Furthermore, we find that the MSE-binding negative regulator Sum1 represses both mORC1 and SMA2 during mitotic growth. Finally, we demonstrate that an MSE deletion strain, which cannot induce mORC1, contains abnormally high Orc1 levels during post-meiotic stages of gametogenesis. Our results reveal the regulatory mechanism that controls mORC1, highlighting a novel developmental stage-specific role for the MSE element in bi-directional mORC1/SMA2 gene activation, and correlating mORC1 induction with declining Orc1 protein levels. Because eukaryotic genes frequently encode multiple transcripts possessing 5′-UTRs of variable length, our results are likely relevant for gene expression during development and disease in higher eukaryotes.

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.

The Enigmatic Conservation of a Rap1 Binding Site in the Saccharomyces cerevisiae HMR-E Silencer

Silencing at the HMR and HML loci in Saccharomyces cerevisiae requires recruitment of Sir proteins to the HML and HMR silencers. The silencers are regulatory sites flanking both loci and consisting of binding sites for the Rap1, Abf1, and ORC proteins, each of which also functions at hundreds of sites throughout the genome in processes unrelated to silencing. Interestingly, the sequence of the binding site for Rap1 at the silencers is distinct from the genome-wide binding profile of Rap1, being a weaker match to the consensus, and indeed is bound with low affinity relative to the consensus sequence. Remarkably, this low-affinity Rap1 binding site variant was conserved among silencers of the sensu stricto Saccharomyces species, maintained as a poor match to the Rap1 genome-wide consensus sequence in all of them. We tested multiple predictions about the possible role of this binding-site variant in silencing by substituting the native Rap1 binding site at the HMR-E silencer with the genome-wide consensus sequence for Rap1. Contrary to the predictions from the current models of Rap1, we found no influence of the Rap1 binding site version on the kinetics of establishing silencing, nor on the maintenance of silencing, nor the extent of silencing. We further explored implications of these findings with regard to prevention of ectopic silencing, and deduced that the selective pressure for the unprecedented conservation of this binding site variant may not be related to silencing.

Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier–Gorlin syndrome

Recognition of distinctly modified histones by specialized “effector” proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes1. Effector proteins influence DNA-templated processes, including transcription, DNA recombination, and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulates DNA replication. Here we show that ORC1 – a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing2 – contains a BAH (bromo adjacent homology) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyllysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at origins, ORC chromatin loading, and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the etiology of Meier-Gorlin syndrome (MGS)3,4, a form of primordial dwarfism5, and ORC1 depletion in zebrafish results in an MGS-like phenotype4. We find that wild-type human ORC1, but not ORC1 H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyllysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal etiologic role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

Trypanosome prereplication machinery: a potential new target for an old problem.

Approximately ten million people suffer from Chagas disease worldwide, caused by Trypanosoma cruzi, with the disease burden predominately focused in Latin America. Sleeping sickness is another serious health problem, caused by Trypanosoma brucei, especially in sub-Saharan countries. Unfortunately, the drugs currently available to treat these diseases have toxic effects and are not effective against all disease phases or parasite strains. Therefore, there is a clear need for the development of novel drugs and drug targets to treat these diseases. We propose the trypanosome prereplication machinery component, Orc1/Cdc6, as a potential target for drug development. In trypanosomes, Orc1/Cdc6 is involved in nuclear DNA replication, and, despite its involvement in such a conserved process, Orc1/Cdc6 is distinct from mammalian Orc1 and Cdc6 proteins. Moreover, RNAi-mediated silencing of trypanosome Orc1/Cdc6 expression in T. brucei decreased cell survival, indicating that Orc1/Cdc6 is critical for trypanosome survival.