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PubMed2001

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

Animals↗
PubMed2024

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Humans↗
PubMed2001

Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins.

BACKGROUND: Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC. RESULTS: The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity. CONCLUSION: Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.

Adenine↗
PubMed1983

Organization of DNA sequences and replication origins at yeast telomeres.

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.

Base Sequence↗
PubMed2006

Characterization of the lymphotropic amplicons-6 and tamplicon-7 vectors derived from HHV-6 and HHV-7.

Amplicon-6 and Tamplicon-7 are novel non-integrating vectors derived from the lymphotropic Human Herpesviruses 6 and 7 (HHV-6 and HHV-7). In the presence of helper viruses the amplicon vectors replicate to yield packaged defective genomes of size approximately 150 kb and consisting of multiple repeat units containing (i) the oriLyt DNA replication origin (ii) the pac-1 and pac-2 cleavage and packaging signals (iii) bacterial plasmid DNA sequences (iv) the chosen transgene(s). Employing CD46 as a receptor HHV-6 gains entry into varied cells, including lymphocytes and dendritic cells, whereas HHV-7 employs the CD4 receptor to target CD4+ cells. The amplicon-based vectors have facilitated the characterization of viral DNA replication and packaging. Following electroporation and helper virus superinfection, the vectors can be transmitted as cell associated and as cell-free virions secreted into the medium. Analyses by flow cytometry have shown good cell spread and efficient gene expression. Exemplary transgenes have included: (i) The Green Fluorescence Protein (GFP) (ii) Genes for potential use in anti-viral vaccination e.g., the HSV-1 glycoprotein D (gD) with and without the trans-membrane region, expressed intracellularly, at the cell membrane or as secreted proteins. (iii) Tumor cell antigens. (iv) Apoptotic genes for development of oncolytic vectors. Due to their cell tropism, their structure as concatemeric genomes, with less than 1.5 kb of viral DNA sequences, the HHV-6 and 7 amplicons have the potential to become unique vectors for immunization and lymphotropic gene therapy.

Gene Amplification↗
PubMed2023

H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins.

The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.

Animals↗
PubMed2003

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin.

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

Adenoviridae↗
PubMed1979

Evidence for a new mechanism of cytotoxicity of 1-beta-D arabinofuranosylcytosine.

Inhibition of DNA synthesis by a pulse of 1-beta-D-arabinofuranosylcytosine (ara-C) results in reinitiation of DNA replication in DNA segments replicated earlier in that S phase and hence double replication of some DNA segments. Experiments were with tissue culture cell lines of human origin. DNA replicated early in S phase was labeled with a pulse of [3H]deoxycytidine with the cells pulsed later in S phase with ara-C. The DNA replicated after the time of the ara-C pulse was density labeled with 5-bromodeoxyuridine. Reinitiation of DNA replication in the already replicated [3H]DNA segments was demonstrated using CsCl density gradient analysis by an increase in the 3H label present in the light-heavy peak of semiconservatively replicated DNA. Also, in DNA of control cells, all of the 3H was in the same strand of the light-heavy DNA duplex as was the 5-bromodeoxyuridine, as shown by alkaline CsCl density gradient analysis of purified light-heavy DNA. However, after a pulse of ara-C, utilization of [3H]DNA strands as template strands was demonstrated by the presence of 3H label at the density of unsubstituted DNA chains in alkaline CsCl gradients of the purified light-heavy DNA. This double replication phenomenon can explain certain chromosomal abnormalities induced by ara-C.

Cell Cycle↗
PubMed1998

[The mechanisms of human cytomegalovirus DNA replication].

Human cytomegalovirus (HCMV) is a human herpesvirus with a linear double stranded DNA which have the largest genomes, about 230 kbp in length, of any of the herpesviruses. The replication of HCMV DNA initiated from HCMV DNA replication origin (oriLyt) and formed concatemers by rolling circule-type replication. The genome inversions of the L and S components occur within concatemeric DNA and formed four isomeres. Then, the concatemeric DNA is cleaved to a unit length at the termini with the "a" sequence as a cis-acting signal and packaged into preformed capsids in the nucleus. Six HCMV genes common to herpesvirus group encode essential proteins that constitute the replication fork complex. In addition, five viral genes are required for efficient DNA replication as trans-acting factors. Several HCMV genes are HSV homologs involved in nucleotide metabolism and DNA repair.

Capsid↗
PubMed1995

Mitochondrial DNA minicircles, lacking replication origins, exist in the cardiac muscle of a young normal subject.

This is the first report that mitochondrial (mt) DNA 'minicircles,' lacking replication origins, exist in the heart mtDNA of a young normal subject. A total detection system using 180 kinds of PCR primer pairs was recently devised to detect all possible deletions in mtDNA. To assess the reliability of the system, template mtDNAs were prepared from the heart muscle by three different methods including DNAase digestion. The methods for mtDNA preparation and the nuclear DNA contaminants were demonstrated to have few influences on the analysis. With not less than 95% accuracy, 52 types of deletions including 36 types of minicircles were detected in the subject's mtDNA. These results will give an insight to the mechanism of mtDNA deletion and to the relevance of the deletion with normal aging.

Adult↗
PubMed2006

Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleoplasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.

Animals↗
PubMed1982

Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: comparisons and evolutionary relationships.

The nucleotide sequences of the Enterobacter aerogenes and Klebsiella pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimurium. Four interrelated, 9-base-pair repeats were identified from the conserved regions within the minimal origin. Evolutionary rates calculated from the minimal origin sequences yielded a quantitative phylogenic tree which agreed with the taxonomic classification of these genera.

Base Sequence↗
PubMed2000

5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates.

Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.

3' Untranslated Regions↗
PubMed1989

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.

Bacteriophage lambda↗
PubMed1997

Cell cycle-dependent establishment of a late replication program.

DNA replication origins in chromosomes of eukaryotes are activated according to a temporal program. In the yeast Saccharomyces cerevisiae, activation of origins in early S phase appears to be a default state. However, cis-acting elements such as telomeres can delay origin activation until late S phase. Site-specific recombination was used to separate origin from telomere in vivo, thereby demonstrating that the signal for late activation is established between mitosis and START in the subsequent G1 phase. Once set, the signal can persist through the next S phase in the absence of the telomere. Establishment of the temporal program and of initiation competence of origins may be coincident events.

DNA Replication↗
PubMed1991

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii.

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.

Ammonium Sulfate↗
PubMed1992

DNA replication and the cell cycle.

The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division. Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells. One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication. RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase. This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication. Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA. A cellular origin binding protein had not been characterized. To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail. This origin consists of four separate functional elements, one of which is essential. A multiprotein complex that binds to the essential element has been identified and purified. This protein complex binds to all known cellular origins from S. cerevisiae and may function as an origin recognition complex.

Animals↗