Molecular evolution of Drosophila Cdc6, an essential DNA replication-licensing gene, suggests an adaptive choice of replication origins

Increased size of eukaryotic genomes necessitated the use of multiple origins of DNA replication, and presumably selected for their efficient spacing to ensure rapid DNA replication. The sequence of these origins remains undetermined in metazoan genomes, leaving important questions about the selective constraints acting on replication origins unanswered. We have chosen to study the evolution of proteins that recognize and define these origins every cell cycle, as a surrogate to the direct analysis of replication origins. Among these DNA replication proteins is the essential Cdc6 protein, which acts to license origins for replication. We find that two different species pairs of Drosophila show evidence of positive selection in Cdc6 in their highly conserved C-terminal AAA-ATPase domain. We also identified amino acid segments that are highly conserved in the N-terminal tail of Cdc6 proteins from various Drosophila species, but are not conserved even in closely related insect species. Instead, we find that the N-terminal tails of Cdc6 proteins vary extensively in size and sequence across different eukaryotic lineages. Our results suggest that choice of origin firing may be significantly altered in closely related species, as each set of replication proteins optimizes to its own genomic landscape.     

The regulatory function of N-terminal AAA+ ATPase domain of eukaryote-like archaeal Orc1/Cdc6 protein during DNA replication initiation

Archaeal replication machinery represents a core version of this in eukaryotes. The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). In this study, we investigate the DNA-binding activities of the N-terminal AAA⁺ ATPase domains of these Orc1/Cdc6 proteins, including their functional interactions with the other SsoCdc6 proteins, on duplex DNA substrates derived from the origins of S. solfataricus. We showed that the ATPase domain of SsoCdc6-2 retained to a great extent the origin DNA-binding activity, and likewise maintained its stimulating effect on SsoCdc6-3. Second, the ATPase domain of SsoCdc6-1, which also stimulated the DNA-binding ability of SsoCdc6-3, demonstrated a significantly improved DNA-binding activity at the forked substrate, but only showed a very weak ability towards the blunt DNA. Third, the ATPase domain of SsoCdc6-3, although having lost much of its DNA-binding activity from the origin, inhibited both SsoCdc6-1 and SsoCdc6-2. These imply that the N-terminal AAA⁺ ATPase domain of archaeal Orc1/Cdc6 protein could be differentially involved in origin recognition during DNA replication initiation even if lacking conventional C-terminal winged helix DNA-binding elements. Our findings further propose that conserved AAA⁺ ATPase domains of Orc1/Cdc6 proteins determine their defined and coordinated functions not only in the archaeon species but also in eukaryotes during the early events of DNA replication.     

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.     

Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture

DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Conservation of epigenetic regulation, ORC binding and developmental timing of DNA replication origins in the genus Drosophila

There is much interest in how DNA replication origins are regulated so that the genome is completely duplicated each cell division cycle and in how the division of cells is spatially and temporally integrated with development. In the Drosophila melanogaster ovary, the cell cycle of somatic follicle cells is modified at precise times in oogenesis. Follicle cells first proliferate via a canonical mitotic division cycle and then enter an endocycle, resulting in their polyploidization. They subsequently enter a specialized amplification phase during which only a few, select origins repeatedly initiate DNA replication, resulting in gene copy number increases at several loci important for eggshell synthesis. Here we investigate the importance of these modified cell cycles for oogenesis by determining whether they have been conserved in evolution. We find that their developmental timing has been strictly conserved among Drosophila species that have been separate for approximately 40 million years of evolution and provide evidence that additional gene loci may be amplified in some species. Further, we find that the acetylation of nucleosomes and Orc2 protein binding at active amplification origins is conserved. Conservation of DNA subsequences within amplification origins from the 12 recently sequenced Drosophila species genomes implicates members of a Myb protein complex in recruiting acetylases to the origin. Our findings suggest that conserved developmental mechanisms integrate egg chamber morphogenesis with cell cycle modifications and the epigenetic regulation of origins.     

Regulating the licensing of DNA replication origins in metazoa

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.     

Four chromosome replication origins in the archaeon Pyrobaculum calidifontis

Replication origins were mapped in hyperthermophilic crenarchaea, using high‐throughput sequencing‐based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb‐1, within the origin. The high‐throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.     

Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix–turn–helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.     

The origin recognition complex protein family

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.     

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.     

Eukaryotic DNA replication: from pre-replication complex to initiation complex

A common mechanism has emerged for the control of the initiation of eukaryotic DNA replication. The minichromosome maintenance protein complex (MCM) and Cdc45 have now been recognized as central components of the initiation machinery. In addition, two types of S phase promoting kinases conserved between yeast and humans play critical roles in the initiation reaction. At the onset of S phase, S phase kinases promote the association of Cdc45 with MCM at origins. Upon the formation of the MCM-Cdc45 complex at origins, the duplex DNA is unwound and various replication proteins, including DNA polymerases, are recruited onto unwound DNA. The increasing number of newly identified factors involved in the initiation reaction indicates that the control of initiation requires highly evolved machinery in eukaryotic cells.     

Eukaryotic origins of DNA replication: could you please be more specific?

Initiation of eukaryotic DNA replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in eukaryotic genomes.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

Drf1, a novel regulatory subunit for human Cdc7 kinase

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.     

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.     

Initiation of eukaryotic DNA replication: conservative or liberal?

The mechanism for initiation of eukaryotic DNA replication is highly conserved: the proteins required to initiate replication, the sequence of events leading to initiation, and the regulation of initiation are remarkably similar throughout the eukaryotic kingdom. Nevertheless, there is a liberal attitude when it comes to selecting initiation sites. Differences appear to exist in the composition of replication origins and in the way proteins recognize these origins. In fact, some multicellular eukaryotes (the metazoans) can change the number and locations of initiation sites during animal development, revealing that selection of initiation sites depends on epigenetic as well as genetic parameters. Here we have attempted to summarize our understanding of this process, to identify the similarities and differences between single cell and multicellular eukaryotes, and to examine the extent to which origin recognition proteins and replication origins have been conserved among eukaryotes. Published 2000 Wiley-Liss, Inc.     

Cdt1 and Cdc6 are destabilized by rereplication-induced DNA damage

The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4(DDB1) ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.