Initiation Sites For DNA Replication Research Articles

Modeling the Dynamics of Eukaryotic DNA Synthesis in Remembrance of Tunde Ogunnaike.

Two things Tunde loved were dynamics and probability. The work described herein combined them both, which explains why Tunde invariably asked me each time we talked how this work was proceeding. However, as I've come to appreciate as reminiscent of a surprisingly large amount of work in almost any researcher's career, I did not complete a peer-reviewed article on the matter while he could see it. We were broadly motivated by analysis of data for total DNA content in single cells, across thousands of cells. From such data one can estimate the proportions of cells in different phases of the cell cycle by fitting a mixture model for subpopulations of G0/G1 phase cells (1 relative copy of the genome), S phase cells (between 1 and 2 relative copies of the genome), and G2/M phase cells (2 relative copies of the genome). Given an asynchronously cycling population, Gaussian models are reasonable for the G0/G1 and G2/M subpopulations, but an appropriate functional form for the S-phase subpopulation was unclear. Since the probability of observing an S-phase cell is intimately related to the dynamics of DNA replication, we worked to derive a model for DNA replication dynamics from first principles, resulting in a closed-form, analytic expression for the dynamics of DNA synthesis. While quite arguably a somewhat superfluous effort, there is a certain satisfaction and academic beauty to modeling systems from a first-principles approach, and it can sometimes lead to unexpected scientific insights. Yet, while mathematically elegant, there was a fundamental issue with a key assumption that the so-called inter-origin distance distribution (distances between DNA replication initiation sites) was time-invariant. First, I present the model as developed previously. Then, to address the time-invariant inter-origin distance distribution issue, I provide a treatment of time-varying inter-origin distance distributions that, while mathematically simple, provides (i) mechanistic predictions for how all the DNA in a fertilized frog egg can be replicated "on time" despite some inter-origin distances initially exceeding the corresponding amount of allowable time and (ii) evidence that, based only on data from DNA content versus time and average inter-origin distances, somatic cell DNA is parsed into distinct regions whose replication is temporally separate.

Bioinformatical dissection of fission yeast DNA replication origins.

Replication origins in eukaryotes form a base for assembly of the pre-replication complex (pre-RC), thereby serving as an initiation site of DNA replication. Characteristics of replication origin vary among species. In fission yeast Schizosaccharomyces pombe, DNA of high AT content is a distinct feature of replication origins; however, it remains to be understood what the general molecular architecture of fission yeast origin is. Here, we performed ChIP-seq mapping of Orc4 and Mcm2, two representative components of the pre-RC, and described the characteristics of their binding sites. The analysis revealed that fission yeast efficient origins are associated with two similar but independent features: a ≥15 bp-long motif with stretches of As and an AT-rich region of a few hundred bp. The A-rich motif was correlated with chromosomal binding of Orc, a DNA-binding component in the pre-RC, whereas the AT-rich region was associated with efficient binding of the DNA replicative helicase Mcm. These two features, in combination with the third feature, a transcription-poor region of approximately 1 kb, enabled to distinguish efficient replication origins from the rest of chromosome arms with high accuracy. This study, hence, provides a model that describes how multiple functional elements specify DNA replication origins in fission yeast genome.

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

Replication origin location might contribute to genetic variability in Trypanosoma cruzi

BackgroundDNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi, the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown.ResultsHere we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1).ConclusionsThese findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins.

Identify origin of replication in Saccharomyces cerevisiae using two-step feature selection technique.

DNA replication is a key step to maintain the continuity of genetic information between parental generation and offspring. The initiation site of DNA replication, also called origin of replication (ORI), plays an extremely important role in the basic biochemical process. Thus, rapidly and effectively identifying the location of ORI in genome will provide key clues for genome analysis. Although biochemical experiments could provide detailed information for ORI, it requires high experimental cost and long experimental period. As good complements to experimental techniques, computational methods could overcome these disadvantages. Thus, in this study, we developed a predictor called iORI-PseKNC2.0 to identify ORIs in the Saccharomyces cerevisiae genome based on sequence information. The PseKNC including 90 physicochemical properties was proposed to formulate ORI and non-ORI samples. In order to improve the accuracy, a two-step feature selection was proposed to exclude redundant and noise information. As a result, the overall success rate of 88.53% was achieved in the 5-fold cross-validation test by using support vector machine. Based on the proposed model, a user-friendly webserver was established and can be freely accessed at http://lin-group.cn/server/iORI-PseKNC2.0. The webserver will provide more convenience to most of wet-experimental scholars.

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome.

R-loops are stable RNA–DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere maintenance, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localization was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the regions between coding sequences of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination the most widespread localization of R-loops in T. brucei suggests a novel correlation with pre-mRNA processing. Finally, we find little evidence for correlation between R-loop localization and mapped sites of DNA replication initiation.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA

Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.

Mapping replication origin sequences in eukaryotic chromosomes.

Recent advances in genome-sequencing technology have led to the complete mapping of DNA replication initiation sites in the human genome. This thorough origin mapping facilitates understanding of the relationship between replication initiation events, transcription, and chromatin modifications, and allows the characterization of consensus sequences of potential replication origins. This unit provides a detailed protocol for isolation and sequence analysis of nascent DNA strands. Two variations of the protocol based on non-overlapping assumptions are described below, addressing potential bias issues for whole-genome analyses.

Enzymatic Rate Enhancements: A Review and Perspective

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.

Serine Hydroxymethyltransferase Anchors de Novo Thymidylate Synthesis Pathway to Nuclear Lamina for DNA Synthesis

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells

Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS), suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation, unveiling a new hallmark in ORI efficiency regulation in mammalian cells.

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.

Isolating Apparently Pure Libraries of Replication Origins from Complex Genomes

Because of the complexity of higher eukaryotic genomes and the lack of a reliable autonomously replicating sequence (ARS) assay for isolating potential replicators, the identification of origins has proven to be extremely challenging and time consuming. We have developed a new origin-trapping method based on the partially circular nature of restriction fragments containing replication bubbles and have prepared a library of approximately 1,000 clones from early S phase CHO cells. When 15 randomly selected clones were analyzed by a stringent two-dimensional (2D) gel replicon mapping method, all were shown to correspond to active, early firing origins. Furthermore, most of these appear to derive from broad zones of potential sites, and the five that were analyzed in a time-course study are all inefficient. This bubble-trapping scheme will allow the construction of comprehensive origin libraries from any complex genome so that their natures and distributions vis-a-vis other chromosomal markers can be established.

Cloning and Characterization of OsORC2, A New Member of Rice Origin Recognition Complex

In eukaryotic cells, the origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. The isolation, characterization and tissue-specific expression of a putative ORC subunit 2 (OsORC2) in Oryza sativa is described here. A novel cDNA fragment encoding rice ORC2 was isolated by screening the subtractive library, which had a higher expression level in inflorescence meristem than in shoot apical meristem. The full-length cDNA of rice ORC2 was obtained by the method of rapid amplification of cDNA ends, which contained an 1140 bp open reading frame encoding a 379 amino acid polypeptide. Sequence alignment shows that there is a high homology between the deduced amino sequence of OsORC2 and maize ORC2 (85%). The tissue-specific expression pattern of OsORC2 reveals that it is abundant in roots, seedling and inflorescence meristem, while its expression level is much lower in mature leaves and shoot.

Studies of the properties of human origin recognition complex and its Walker A motif mutants

The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. In this report we describe the isolation of the wild-type Homo sapiens (Hs)ORC and variants containing a Walker A motif mutation in the Orc1, Orc4, or Orc5 subunit using the baculovirus-expression system. Coexpression of all six HsORC subunits yielded a stable complex containing HsOrc subunits 1-5 (HsORC1-5) with virtually no Orc6 protein (Orc6p). We examined the ATPase, DNA-binding, and replication activities of these complexes. Similar to other eukaryotic ORCs, wild-type HsORC1-5 possesses ATPase activity that is stimulated only 2-fold by single-stranded DNA. HsORC1-5 with a mutated Walker A motif in Orc1p contains no ATPase activity, whereas a similar mutation of either the Orc4 or Orc5 subunit did not affect this activity. The DNA-binding activity of HsORC1-5, using lamin B2 DNA as substrate, is stimulated by ATP 3- to 5-fold. Mutations in the Walker A motif of Orc1p, Orc4p, or Orc5p reduced the binding efficiency of HsORC1-5 modestly (2- to 5-fold). Xenopus laevis ORC-depleted extracts supplemented with HsORC1-5 supported prereplication complex formation and X. laevis sperm DNA replication, whereas the complex with a mutation in the Walker A motif of the Orc1, Orc4, or Orc5 subunit did not. These studies indicate that the ATP-binding motifs of Orc1, Orc4, and Orc5 are all essential for the replication activity associated with HsORC.

Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene

Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1–1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene.

Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator

Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes. We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae. In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1. This analysis applied to a 1–1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region. With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region. This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.

Immortalization of human WI38 cells is associated with differential activation of the c-myc origins.

To study the possible relationships between origin activities and cellular processes leading to malignancy, we used an isogenic system of human embryo lung fibroblast cells WI38 and a SV40-transformed variant, WI38 VA13 2RA (WI38(SV40)). We found that the activities of all initiation sites at the c-myc locus were approximately two-fold as high in WI38(SV40) cells as in WI38 cells. Thus, higher initiation frequency of origins at certain loci is induced with cell immortalization, one of the steps in the multi-step process leading to malignancy. We measured the activities of the four c-myc promoters P0, P1, P2, and P3 with nuclear runon assay in the two cell lines in order to detect potential individual promoter changes that may be also associated with immortalization by SV40 virus. The results show that the activities of the promoters P0, P1, and P3 did not significantly change, but the activity of the major promoter P2 in WI38(SV40) cells was about 7.5- to 8.0-fold as high as that in WI38 cells. The increased activity of promoter P2, although approximately 600 bp downstream of one of the major DNA replication initiation sites, had no preferential influence on the major sites of origin activity. Since the distribution of nascent strand abundance was not significantly altered, binding of transcription factors does not seem to facilitate the assembly of pre-replication complex (pre-RC) or otherwise preferentially alter the activities of the DNA replication proteins at this major initiation site.