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BMC Research Notes Jul 2021The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin...
OBJECTIVE
The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study.
RESULTS
Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.
Topics: Archaea; DNA Replication; DNA, Single-Stranded; Origin Recognition Complex; Protein Binding; Replication Origin
PubMed: 34281605
DOI: 10.1186/s13104-021-05690-w -
Proceedings of the National Academy of... May 2023coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of...
coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters and compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.
Topics: Cell Cycle; DNA Replication; Escherichia coli; Chromosomes; Adenosine Triphosphate
PubMed: 37220276
DOI: 10.1073/pnas.2213795120 -
Nucleic Acids Research May 2002We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in...
We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G1 phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G1 phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G1 phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.
Topics: Antibodies, Monoclonal; Cell Cycle; Cell Nucleus; Cell-Free System; DNA Replication; DNA, Neoplasm; Deoxyuridine; HeLa Cells; Humans; Idoxuridine; Lamin Type B; Lamins; Microscopy, Confocal; Microscopy, Fluorescence; Mimosine; Nuclear Proteins; S Phase; Tumor Cells, Cultured
PubMed: 12000831
DOI: 10.1093/nar/30.10.2114 -
Microbiology and Molecular Biology... Jun 1998An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used... (Review)
Review
An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.
Topics: Base Sequence; DNA Replication; DNA, Bacterial; Leucine Zippers; Models, Genetic; Molecular Sequence Data; Plasmids; RNA, Antisense; Sequence Alignment
PubMed: 9618448
DOI: 10.1128/MMBR.62.2.434-464.1998 -
International Journal of Molecular... Jun 2023Origins of replication are genomic regions in which replication initiates in a bidirectional manner. Recently, a new methodology (origin-derived single-stranded DNA...
Origins of replication are genomic regions in which replication initiates in a bidirectional manner. Recently, a new methodology (origin-derived single-stranded DNA sequencing; ori-SSDS) was developed that allows the detection of replication initiation in a strand-specific manner. Reanalysis of the strand-specific data revealed that 18-33% of the peaks are non-symmetrical, suggesting a single direction of replication. Analysis of replication fork direction data revealed that these are origins of replication in which the replication is paused in one of the directions, probably due to the existence of a replication fork barrier. Analysis of the unidirectional origins revealed a preference of G4 quadruplexes for the blocked leading strand. Taken together, our analysis identified hundreds of genomic locations in which the replication initiates only in one direction, and suggests that G4 quadruplexes may serve as replication fork barriers in such places.
Topics: Animals; Mice; DNA Replication; DNA, Single-Stranded; Replication Origin
PubMed: 37298562
DOI: 10.3390/ijms24119611 -
Nucleic Acids Research Dec 2016Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins,...
Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.
Topics: Cell Line, Tumor; Chromosome Mapping; Computational Biology; DNA; DNA Replication; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Nucleotide Motifs; Replication Origin; Sequence Analysis, DNA; Transcription Initiation Site
PubMed: 27587586
DOI: 10.1093/nar/gkw760 -
Biochemical Society Transactions Feb 2019It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis Rather, they require... (Review)
Review
It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis Rather, they require a 3'-OH group from which to extend a DNA chain. Cellular DNA replication systems exploit a dedicated, limited processivity RNA polymerase, termed primase, that synthesizes a short oligoribonucleotide primer which is then extended by a DNA polymerase. Thus, primases can initiate synthesis, proceed with primer elongation for a short distance then transfer the primer to a DNA polymerase. Despite these well-established properties, the mechanistic basis of these dynamic behaviours has only recently been established. In the following, the author will describe recent insights from studies of the related eukaryotic and archaeal DNA primases. Significantly, the general conclusions from these studies likely extend to a broad class of extrachromosomal element-associated primases as well as the human primase-related DNA repair enzyme, PrimPol.
Topics: Archaeal Proteins; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes
PubMed: 30647143
DOI: 10.1042/BST20180627 -
Genes To Cells : Devoted To Molecular &... Jun 2002To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that... (Review)
Review
To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Its control has been uncovered by the discovery of the CDKs (cyclin dependent kinases) as master regulators of the cell cycle and the initiator proteins of DNA replication, such as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At the end of mitosis, the MCM complex is loaded on to chromatin with the aid of ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs, together with the Cdc7 kinase, trigger the initiation of replication, recruiting the DNA replicating enzymes on sites of replication. The activated MCM complex appears to play a key role in the DNA unwinding step, acting as a replicating helicase and moves along with the replication fork, at the same time bringing the origins to the unlicensed state. The cycling of CDK activity in the cell cycle separates the two states of replication origins, the licensed state in G1-phase and the unlicensed state for the rest of the cell cycle. Only when CDK drops at the completion of mitosis, is the restriction on licensing relieved and a new round of replication is allowed. Such a CDK-regulated licensing control is conserved from yeast to higher eukaryotes, and ensures that DNA replication takes place only once in a cycle. Xenopus laevis and mammalian cells have an additional system to control licensing. Geminin, whose degradation at the end of mitosis is essential for a new round of licensing, has been shown to bind Cdt1 and negatively regulate it, providing a new insight into the regulation of DNA replication in higher eukaryotes.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinases; DNA Replication; DNA-Binding Proteins; Geminin; Gene Expression Regulation; Humans; Origin Recognition Complex; Replication Origin; Saccharomyces cerevisiae Proteins; Xenopus; Xenopus Proteins; Yeasts
PubMed: 12059957
DOI: 10.1046/j.1365-2443.2002.00544.x -
Genes & Development Jun 2010Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively... (Review)
Review
Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively poorly. Two conserved kinases called Cdc7 (cell division cycle 7) and cyclin-dependent kinase (CDK) are required to establish replication forks during the initiation of chromosome replication, and a key feature of this process is the activation of the replicative DNA helicase in situ at each origin of DNA replication. A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.
Topics: Animals; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes; Cyclin-Dependent Kinases; DNA Replication; Eukaryotic Cells; Humans; Protein Serine-Threonine Kinases; RecQ Helicases; Replication Origin; S Phase; Saccharomyces cerevisiae; Schizosaccharomyces
PubMed: 20551170
DOI: 10.1101/gad.1933010 -
Seminars in Cancer Biology Apr 2013Normal cellular division requires that the genome be faithfully replicated to ensure that unaltered genomic information is passed from one generation to the next. DNA... (Review)
Review
Normal cellular division requires that the genome be faithfully replicated to ensure that unaltered genomic information is passed from one generation to the next. DNA replication initiates from thousands of origins scattered throughout the genome every cell cycle; however, not all origins initiate replication at the same time. A vast amount of work over the years indicates that different origins along each eukaryotic chromosome are activated in early, middle or late S phase. This temporal control of DNA replication is referred to as the replication-timing program. The replication-timing program represents a very stable epigenetic feature of chromosomes. Recent evidence has indicated that the replication-timing program can influence the spatial distribution of mutagenic events such that certain regions of the genome experience increased spontaneous mutagenesis compared to surrounding regions. This influence has helped shape the genomes of humans and other multicellular organisms and can affect the distribution of mutations in somatic cells. It is also becoming clear that the replication-timing program is deregulated in many disease states, including cancer. Aberrant DNA replication timing is associated with changes in gene expression, changes in epigenetic modifications and an increased frequency of structural rearrangements. Furthermore, certain replication timing changes can directly lead to overt genomic instability and may explain unique mutational signatures that are present in cells that have undergone the recently described processes of "chromothripsis" and "kataegis". In this review, we will discuss how the normal replication timing program, as well as how alterations to this program, can contribute to the evolution of the genomic landscape in normal and cancerous cells.
Topics: Animals; DNA Replication; DNA Replication Timing; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Models, Biological; Neoplasms; Time Factors
PubMed: 23327985
DOI: 10.1016/j.semcancer.2013.01.001