Chromosomal Replication Origin Research Articles

Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Replication of eukaryotic genomes is limited to once per cell cycle, by a two-step mechanism. DNA replication origins are first "licensed" during G1 phase by loading of an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). Initiation then occurs during S phase, triggered by cyclin-dependent kinases (CDKs), which promote recruitment of proteins required for helicase activation and replisome assembly. CDKs and the anaphase promoting complex/cyclosome (APC/C) restrict licensing to G1 phase by directly and indirectly regulating pre-RC components, including ORC, Cdc6, Cdt1, and Mcm2-7. Despite the fundamental importance of licensing regulation, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. Here we show that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: eliminating any single pre-RC-regulatory mechanism has very little affect on viability. The second is interchangeability: we show that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. These experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome-replication cycle.

Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions

Eukaryotic DNA replication requires the assembly of multiprotein pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive complex. In the second step, the salt-sensitive complex is slowly converted to a salt-stable complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA complexes contain underwound or negatively writhed DNA. Purified human origin recognition complex (ORC) induces similar topological changes in origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA complex and enhance the SpORC-dependent topology changes in origin DNA. Thus, the formation of stable protein-DNA complexes at S. pombe origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings demonstrate that SpORC is not the sole determinant of origin recognition.

Dynamics of genome rearrangement in bacterial populations.

Genome structure variation has profound impacts on phenotype in organisms ranging from microbes to humans, yet little is known about how natural selection acts on genome arrangement. Pathogenic bacteria such as Yersinia pestis, which causes bubonic and pneumonic plague, often exhibit a high degree of genomic rearrangement. The recent availability of several Yersinia genomes offers an unprecedented opportunity to study the evolution of genome structure and arrangement. We introduce a set of statistical methods to study patterns of rearrangement in circular chromosomes and apply them to the Yersinia. We constructed a multiple alignment of eight Yersinia genomes using Mauve software to identify 78 conserved segments that are internally free from genome rearrangement. Based on the alignment, we applied Bayesian statistical methods to infer the phylogenetic inversion history of Yersinia. The sampling of genome arrangement reconstructions contains seven parsimonious tree topologies, each having different histories of 79 inversions. Topologies with a greater number of inversions also exist, but were sampled less frequently. The inversion phylogenies agree with results suggested by SNP patterns. We then analyzed reconstructed inversion histories to identify patterns of rearrangement. We confirm an over-representation of “symmetric inversions”—inversions with endpoints that are equally distant from the origin of chromosomal replication. Ancestral genome arrangements demonstrate moderate preference for replichore balance in Yersinia. We found that all inversions are shorter than expected under a neutral model, whereas inversions acting within a single replichore are much shorter than expected. We also found evidence for a canonical configuration of the origin and terminus of replication. Finally, breakpoint reuse analysis reveals that inversions with endpoints proximal to the origin of DNA replication are nearly three times more frequent. Our findings represent the first characterization of genome arrangement evolution in a bacterial population evolving outside laboratory conditions. Insight into the process of genomic rearrangement may further the understanding of pathogen population dynamics and selection on the architecture of circular bacterial chromosomes.

Schizosaccharomyces pombe Noc3 Is Essential for Ribosome Biogenesis and Cell Division but Not DNA Replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00 degrees values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Par genes in Mycobacterium bovis and Mycobacterium smegmatis are arranged in an operon transcribed from "SigGC" promoters.

BackgroundThe ParA/Soj and ParB/Spo0J proteins, and the cis-acting parS site, participate actively in chromosome segregation and cell cycle progression. Genes homologous to parA and parB, and two putative parS copies, have been identified in the Mycobacterium bovis BCG and Mycobacterium smegmatis chromosomes. As in Mycobacterium tuberculosis, the parA and parB genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. The present work focused on the determination of the transcriptional organisation of the ~6 Kb orf60K-parB region of M. bovis BCG and M. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein (GFP) and quantitative RT-PCR.ResultsThe parAB genes were arranged in an operon. However, we also found promoters upstream of each one of these genes. Seven putative promoter sequences were identified in the orf60K-parB region of M. bovis BCG, whilst four were identified in the homologous region of M. smegmatis, one upstream of each open reading frame (ORF).Real-time PCR assays showed that in M. smegmatis, mRNA-parA and mRNA-parB levels decreased between the exponential and stationary phases. In M. bovis BCG, mRNA-parA levels also decreased between the exponential and stationary phases. However, parB expression was higher than parA expression and remained almost unchanged along the growth curve.ConclusionThe majority of the proposed promoter regions had features characteristic of Mycobacterium promoters previously denoted as Group D. The -10 hexamer of a strong E. coli σ70-like promoter, located upstream of gidB of M. bovis BCG, overlapped with a putative parS sequence, suggesting that the transcription from this promoter might be regulated by the binding of ParB to parS.

Roles for the AAA+ motifs of DnaA in the initiation of DNA replication

The cell-cycle-co-ordinated initiation of chromosomal replication is highly regulated. The ordered assembly and conformational change of specific proteins at the replication origin are crucial to the process of replication initiation. In Escherichia coli, ATP-DnaA molecules form multimeric complexes with the chromosomal origin of replication (oriC), and unwind the duplex DNA within oriC, resulting in initiation of replication. DnaA is a common protein in bacterial species and plays a main and crucial role in the initiation of chromosomal replication. Unlike well-characterized AAA+ (ATPase associated with various cellular activities) proteins such as chaperons and proteases, DnaA molecules stably take on a monomeric form and form homomultimers in a manner dependent on binding to oriC. The oriC region carries several DnaA-binding sites with various affinities. Recent progress in the analysis of DnaA and related proteins has revealed specific roles for the AAA+ unique motifs of DnaA. These results suggest mechanisms for recognition of ATP bound to DnaA, the co-operative binding of ATP-DnaA molecules on oriC, the formation of an ATP-DnaA-specific oriC complex, an initiation complex and regulatory hydrolysis of DnaA-bound ATP.

The Genome Sequence of the Tomato-Pathogenic ActinomyceteClavibacter michiganensissubsp.michiganensisNCPPB382 Reveals a Large Island Involved in Pathogenicity

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.

DNA mismatch repair-induced double-strand breaks

Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis (PFGE), that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O 6-methylguanine (O 6-meG) base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.

Hexameric ring structure of human MCM10 DNA replication factor

The DNA replication factor minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein crucial for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes, MCM10 recruitment to replication origins is required to provide a physical link between the MCM2-7 complex DNA helicase and DNA polymerases. Here, we report the molecular structure of human MCM10 as determined by electron microscopy and single-particle analysis. The MCM10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. This structure, together with biochemical data, suggests that this important protein uses its architecture to provide a docking module for assembly of the molecular machinery required for eukaryotic DNA replication.

Structure and Function of DnaA N-terminal Domains: SPECIFIC SITES AND MECHANISMS IN INTER-DnaA INTERACTION AND IN DnaB HELICASE LOADING ON oriC

DnaA forms a homomultimeric complex with the origin of chromosomal replication (oriC) to unwind duplex DNA. The interaction of the DnaA N terminus with the DnaB helicase is crucial for the loading of DnaB onto the unwound region. Here, we determined the DnaA N terminus structure using NMR. This region (residues 1–108) consists of a rigid region (domain I) and a flexible region (domain II). Domain I has an α-α-β-β-α-β motif, similar to that of the K homology (KH) domain, and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggest a model for DnaA homomultimer formation and DnaB helicase loading on oriC.

Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes

In all three domains of life, DNA replication begins at specialized loci termed replication origins. In bacteria, replication initiates from a single, clearly defined site. In contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. Recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon Sulfolobus has three replication origins. However, the evolutionary mechanism driving the progression from single to multiple origin usage remains unclear. Here, we demonstrate that Aeropyrum pernix, a distant relative of Sulfolobus, has two origins. Comparison with the Sulfolobus origins provides evidence for evolution of replicon complexity by capture of extrachromosomal genetic elements. We additionally identify a previously unrecognized candidate archaeal initiator protein that is distantly related to eukaryotic Cdt1. Our data thus provide evidence that horizontal gene transfer, in addition to its well-established role in contributing to the information content of chromosomes, may fundamentally alter the manner in which the host chromosome is replicated.

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

Perturbation of the Activity of Replication Origin by Meiosis-specific Transcription

We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in premeiotic S-phase by neutral/neutral two-dimensional gel electrophoresis. The comparison of origin activity of each origin in mitotic and premeiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in premeiotic S-phase. ARS605 is located within the open reading frame of MSH4 gene that is transcribed specifically during an early stage of meiosis. Systematic analysis of relationships between MSH4 transcription and ARS605 origin activity revealed that transcription of MSH4 inhibited the ARS605 origin activity by removing origin recognition complex from ARS605. Deletion of UME6, a transcription factor responsible for repressing MSH4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.

Tools for the genetic analysis of Mycoplasma

The mycoplasmas have attracted much scientific attention since they contain the smallest known genomes of any independently viable bacterial species. However, the detailed genetic analysis of these bacteria has lagged behind the well-analyzed bacterial model organisms for a long time. This is due to the use of the UGA codon for tryptophan rather than as stop codon, which had often prevented the expression of full-length Mycoplasma proteins in heterologous hosts. Additionally, insufficient efficiency of homologous recombination prevented the targeted disruption of genes in some species such as M. pneumoniae whereas homologous recombination is operative in other mycoplasmas. Only recently, efficient screening systems for the use of transposon-based mutagenesis have been developed to circumvent this problem and to allow the targeted isolation of desired transposon insertion mutants. With the availability of several Mycoplasma genome sequences, artificial plasmids based on the chromosomal origin of replication were constructed that can now be used for complementation studies and for the stable introduction of foreign genetic material. In this review, we give an overview on recent developments in Mycoplasma genetics that facilitate the genetic manipulation of these interesting organisms.

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites

In bacteria, Ter sites bound to Tus/Rtp proteins halt replication forks moving only in one direction, providing a convenient mechanism to terminate them once the chromosome had been replicated. Considering the importance of replication termination and its position as a checkpoint in cell division, the accumulated knowledge on these systems has not dispelled fundamental questions regarding its role in cell biology: why are there so many copies of Ter, why are they distributed over such a large portion of the chromosome, why is the tus gene not conserved among bacteria, and why do tus mutants lack measurable phenotypes? Here we examine bacterial genomes using bioinformatics techniques to identify the region(s) where DNA polymerase III-mediated replication has historically been terminated. We find that in both Escherichia coli and Bacillus subtilis, changes in mutational bias patterns indicate that replication termination most likely occurs at or near the dif site. More importantly, there is no evidence from mutational bias signatures that replication forks originating at oriC have terminated at Ter sites. We propose that Ter sites participate in halting replication forks originating from DNA repair events, and not those originating at the chromosomal origin of replication.

Binding of AlF-C, an Orc1-Binding Transcriptional Regulator, Enhances Replicator Activity of the Rat Aldolase B Origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.

DNA elements modulating the KARS12 chromosomal replicator in Kluyveromyces lactis

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.

Bacterial Division: Another Way to Box in the Ring

Proper placement of the cell division site in some rod-shaped bacteria requires two different negative regulatory systems, nucleoid occlusion and the Min proteins. Caulobacter crescentus lacks these systems, but recent work has uncovered a novel regulator that achieves the same goals.

Phylogenetic analysis of enteric species of the family Enterobacteriaceae using the oriC-locus

Phylogenetic analysis of 38 enteric species belonging to the Enterobacteraceae family was carried out using the non-coding locus oriC, the chromosomal replication origin. The oriC loci were amplified with conserved oligonucleotides and the PCR fragments were sequenced directly. The results establish a phylogenetic tree for the classification of different species of the genera Escherichia, Shigella, Salmonella, Enterobacter, Citrobacter, Klebsiella, Raoultella, Kluyvera, Cedecea and Buttiauxella. Functional important protein-binding sites located in oriC are well conserved throughout the enteric group. More over, due to a high overall divergence value phylogenetic trees were robust and well supported by bootstrap analysis. In comparison with 16S rDNA analysis, the oriC sequences indicated a greater evolutionary divergence for bacteria. We propose that the oriC locus might be a suitable phylogenetic marker for the identification and classification of bacteria, in particular for closely related species.