Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly.

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

An Orc1/Cdc6 ortholog functions as a key regulator in the DNA damage response in Archaea.

While bacteria and eukaryotes show distinct mechanisms of DNA damage response (DDR) regulation, investigation of ultraviolet (UV)-responsive expression in a few archaea did not yield any conclusive evidence for an archaeal DDR regulatory network. Nevertheless, expression of Orc1-2, an ortholog of the archaeal origin recognition complex 1/cell division control protein 6 (Orc1/Cdc6) superfamily proteins was strongly activated in Sulfolobus solfataricus and Sulfolobus acidocaldarius upon UV irradiation. Here, a series of experiments were conducted to investigate the possible functions of Orc1-2 in DNA damage repair in Sulfolobus islandicus. Study of DDR in Δorc1-2 revealed that Orc1-2 deficiency abolishes DNA damage-induced differential expression of a large number of genes and the mutant showed hypersensitivity to DNA damage treatment. Reporter gene and DNase I footprinting assays demonstrated that Orc1-2 interacts with a conserved hexanucleotide motif present in several DDR gene promoters and regulates their expression. Manipulation of orc1-2 expression by promoter substitution in this archaeon revealed that a high level of orc1-2 expression is essential but not sufficient to trigger DDR. Together, these results have placed Orc1-2 in the heart of the archaeal DDR regulation, and the resulting Orc1-2-centered regulatory circuit represents the first DDR network identified in Archaea, the third domain of life.

Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli.

The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling.

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation.

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.

Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner.

In eukaryotes, origin recognition complex (ORC) proteins establish the pre-replicative complex (preRC) at the origins, and this is essential for the initiation of DNA replication. Open chromatin structures regulate the efficiency of preRC formation and replication initiation. However, the molecular mechanisms that control chromatin structure, and how the preRC components establish themselves on the chromatin remain to be understood. In human cells, the ORC is a highly dynamic complex with many separate functions attributed to sub-complexes or individual subunits of the ORC, including heterochromatin organization, telomere and centromere function, centrosome duplication and cytokinesis. We demonstrate that human Orc5, unlike other ORC subunits, when ectopically tethered to a chromatin locus, induces large-scale chromatin decondensation, predominantly during G1 phase of the cell cycle. Orc5 associates with the H3 histone acetyl transferase GCN5 (also known as KAT2A), and this association enhances the chromatin-opening function of Orc5. In the absence of Orc5, histone H3 acetylation is decreased at the origins. We propose that the ability of Orc5 to induce chromatin unfolding during G1 allows the establishment of the preRC at the origins.

Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.

Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs.

An orc1 allele with a mutated APC motif is female sterile with amplification defects.

The origin recognition complex 1 (ORC1) is the largest subunit of the ORC, the heteromeric hexamer. ORC1 is an essential component of the pre-replicative complex (pre-RC) that licenses eukaryote DNA replication origins. The levels of ORC1 fluctuate during the mitotic cell cycle in Drosophila as well as in some human cells. Proteolysis of ORC1 occurs at the end of M phase in Drosophila, which is mediated by the anaphase-promoting complex (APC), and in late S phase in human cells by Skip-Cullin-F box (SCF). Previously we showed that proteolysis of ORC1 by APC is mediated by the ORC1 destruction box (the O-box), an APC motif conserved among species yet distinct from the D-box or KEN-box. Recently we showed that replacing the O-box with the D-box (ORC1O→D) changes the degradation profile of ORC1 during a canonical cell cycle. Here we report further characterization of the ORC1O→D allele that turned out to be a useful tool to examine the function of ORC1 in other modes of DNA replication during oogenesis. In endoreplication stages ORC1O→D does not change any DNA content profiles, consistent with our previous finding that ORC is dispensable for endoreplication. However, in amplification stage replication efficiency of ORC1O→D is drastically reduced, which resulted in amplification defects that led to thin egg shell phenotype. Taken together, our analyses show that orc1 allele newly identified is female sterile and possesses a unique feature of phenotypes that are distinct in different modes of DNA replication.

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing.

Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N(1-238)) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N(1-238), endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N(1-238) compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum.

Linking DNA replication to heterochromatin silencing and epigenetic inheritance.

Chromatin is organized into distinct functional domains. During mitotic cell division, both genetic information encoded in DNA sequence and epigenetic information embedded in chromatin structure must be faithfully duplicated. The inheritance of epigenetic states is critical in maintaining the genome integrity and gene expression state. In this review, we will discuss recent progress on how proteins known to be involved in DNA replication and DNA replication-coupled nucleosome assembly impact on the inheritance and maintenance of heterochromatin, a tightly compact chromatin structure that silences gene transcription. As heterochromatin is important in regulating gene expression and maintaining genome stability, understanding how heterochromatin states are inherited during S phase of the cell cycle is of fundamental importance.

Function of the origin recognition complex 1 (ORC1) outside DNA replication in Drosophila.

The origin recognition complex (ORC) is an essential component of the pre-replicative complex (pre-RC) that binds to replication origins for licensing. Levels of the largest ORC subunit, ORC1, oscillate during the mitotic cell cycle and regulate origin usage. In Drosophila, ORC1 levels increase at the G(1)/S transition following E2F-dependent transcriptional activation, remain high until the end of M phase and then decrease at the M/G(1) transition when ORC1 is targeted for proteolysis by the anaphase-promoting complex (APC). A function, if any, for Drosophila ORC1 after S phase has not been described. Here, we determined the role of ORC1 at stages outside S phase by generating ORC1 derivatives with a modified ORC1 degradation box (the O-box) and examining the effects in vivo. These modifications either stabilized ORC1 by mutating the O-box (ORC1(Omut)) so that it is no longer targeted by APC or changed its degradation profile by replacing the O-box with the D-box of human cyclin B (ORC1(O→D)), so that degradation would occur earlier. We determined the distribution and tested the function of these ORC1 derivatives in an orc1 mutant background so that only the mutated protein was expressed. Stable version of ORC1, ORC1 (Omut), showed no effects on cell cycle progression; however, ORC1(O→D), which is degraded early at the G(2)/M transition, led to a higher frequency of M-phase cells but not S-phase cells. Taken together, our results indicate the timing of ORC1 degradation is required for timely progression in M phase.

Radial glia regulate Cajal-Retzius cell positioning in the early embryonic cerebral cortex.

The organization of neocortex, along its radial axis, into a six-layered structure is one of the most exquisite features of the brain. Because of their strategic localization in the marginal zone, and their expression of reelin, a signal that controls spatial ordering of cortical layers, Cajal-Retzius (C-R) cells play a crucial role in cortical patterning along this axis. Yet, it remains less well understood how C-R cell targeting itself is regulated. At the onset of corticogenesis when C-R cells first arrive in the cortex via tangential migration, radial glia (RG) are the main cell type present. This suggests that RG may play a role in C-R cell localization. To test this, we used genetic approaches to perturb RG scaffold during early corticogenesis. We found that disrupting RG endfoot adhesion to basal lamina consistently results in C-R cell displacement. These displacements do not appear to result from primary defects in neural progenitor cell proliferation, deficits in the meninges or basement membrane, or cell autonomous defects in C-R cells. Instead, they show close temporal and spatial correlation with RG endfoot retraction. Moreover, ablation of RG via cell cycle blockade similarly results in local displacement of C-R cells. These lines of evidence thus indicate that, during early corticogenesis, RG play a primary role in regulating spatial targeting of C-R cells. Since RG are also neural progenitors as well as neuronal migration scaffolds, these findings suggest that, during nervous system development, neuroepithelial stem cells may not only be responsible for generating a diverse array of neuronal cell types and facilitating their radial migration. They may also, through regulating the placement of guidepost cells, coordinate spatial patterning of the nervous system along its radial axis.

Same partners, different dance: involvement of DNA replication proteins in centrosome regulation.

Genome replication is the most fundamental element of the continuity of life. In eukaryotes, DNA replication is regulated by an elegant network of many different protein factors to ensure the timely and accurate copying of their entire genome once per cell cycle. The replication factors include the maintenance (MCM) proteins, Cdt1, Cdc6, Cdc7, Cdc45, and geminin. All of these proteins are involved in the regulation of DNA replication at the initiation step. Interestingly, recent studies have shown that some of these replication proteins also localize to the centrosome, often throughout the entire cell cycle. These centrosomally localized replication proteins appear to play essential roles in the regulation of centrosome biogenesis, suggesting that genome replication and segregation are regulated interdependently. In this review, we summarize and discuss the inter-dependent regulation played by some of the replication proteins.

The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication.

Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication. Cyclin A-Cdk2 localizes on centrosomes by means of a modular centrosomal localization sequence (CLS) that is distinct from that of cyclin E. Here, we show that cyclin A interacts with both MCM5 and Orc1 in a CLS-dependent but Cdk-independent manner. Although the MRAIL hydrophobic patch is contained within the cyclin A CLS, binding of both MCM5 and Orc1 to cyclin A does not require a wild-type hydrophobic patch. The same domain in MCM5 that mediates interaction with cyclin E also binds cyclin A, resulting in centrosomal localization of MCM5. Finally, unlike its function in DNA synthesis, MCM5-mediated inhibition of centrosome reduplication in S-phase-arrested CHO cells does not require binding to other MCM family members. These results suggest that cyclins E and A sequentially prevent centrosome reduplication throughout interphase by recruitment of DNA replication factors such as MCM5 and Orc1.

Novel insights into the plant histone code: lessons from ORC1.

Gene expression control depends on the combinatorial presence of various posttranslational modifications of the N-terminal tail of histones, among which acetylation and methylation of lysine residues are the most conspicuous. Effector proteins have been identified that recognize specific histone modifications and transduce the information of the histone code into different degrees of chromatin compaction in either facultative or constitutive heterochromatin. In addition, they also impinge on transcriptional control, either activation or repression, of euchromatic genes. However, a large variety of effector proteins lead to different gene expression readouts. This is complicated by accumulating evidence showing that the consequences of various histone modifications differ among animals and plants. Given the conservation of histone marks, and histone modification enzymes, this has evolutionary implications as to how they are interpreted differently in different organisms. One example that illustrates such diversity of the histone code is the large subunit of the origin recognition complex, ORC1. In plants, but not in yeast and animal cells, ORC1 functions as a transcriptional activator of a subset of target genes and is a plant homeodomain (PHD)-containing protein that binds to histone H3K4me3 residues.

Functional analysis of an Orc6 mutant in Drosophila.

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G(1) and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G(1) arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.

Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions.

Archaeal DNA replication machinery represents a core version of that found in eukaryotes. However, the proteins essential for the coordination of origin selection and the functioning of DNA polymerase have not yet been characterized in archaea, and they are still being investigated in eukaryotes. In the current study, the Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus solfataricus were found to physically interact with its DNA polymerase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the DNA-binding ability of SsoPolB1 and differentially regulated both its polymerase and nuclease activities. Furthermore, the proteins also mutually regulated their interactions with SsoPolB1. In addition, SsoPolB1c467, a nuclease domain-deleted mutant of SsoPolB1 defective in DNA binding, retains the ability to physically interact with SsoCdc6 proteins. Its DNA polymerase activity could be stimulated by these proteins. We report on a linkage between the initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. Our present and previous findings indicate that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection and cell-cycle control of replication.

Arabidopsis ORC1 is a PHD-containing H3K4me3 effector that regulates transcription.

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom.

Elaboration, diversification and regulation of the Sir1 family of silencing proteins in Saccharomyces.

Heterochromatin renders domains of chromosomes transcriptionally silent and, due to clonal variation in its formation, can generate heritably distinct populations of genetically identical cells. Saccharomyces cerevisiae's Sir1 functions primarily in the establishment, but not the maintenance, of heterochromatic silencing at the HMR and HML loci. In several Saccharomyces species, we discovered multiple paralogs of Sir1, called Kos1-Kos4 (Kin of Sir1). The Kos and Sir1 proteins contributed partially overlapping functions to silencing of both cryptic mating loci in S. bayanus. Mutants of these paralogs reduced silencing at HML more than at HMR. Most genes of the SIR1 family were located near telomeres, and at least one paralog was regulated by telomere position effect. In S. cerevisiae, Sir1 is recruited to the silencers at HML and HMR via its ORC interacting region (OIR), which binds the bromo adjacent homology (BAH) domain of Orc1. Zygosaccharomyces rouxii, which diverged from Saccharomyces after the appearance of the silent mating cassettes, but before the whole-genome duplication, contained an ortholog of Kos3 that was apparently the archetypal member of the family, with only one OIR. In contrast, a duplication of this domain was present in all orthologs of Sir1, Kos1, Kos2, and Kos4. We propose that the functional specialization of Sir3, itself a paralog of Orc1, as a silencing protein was facilitated by the tandem duplication of the OIR domain in the Sir1 family, allowing distinct Sir1-Sir3 and Sir1-Orc1 interactions through OIR-BAH domain interactions.