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Atrial myocytes cultured from explanted human adult myocardium demonstrate DNA replication.

The current theory of myocardial development holds that after a limited number of divisions, the myocardiocytes of the developing heart are irreversibly withdrawn from the generation cycle. It is, therefore, considered impossible to grow adult human myocardiocytes in culture, making it necessary for studies of cardiac muscle in culture to be carried out using animal or fetal human models. Recently, we developed a method for isolating, culturing, and characterizing myocardiocytes derived from explanted adult human atrial myocardium. A highly pure fraction (93%) of one of four morphologically discrete cell populations was separated using selective attachment techniques. These cells possessed features consistent with those seen in animal and fetal myocardiocytes. Using immunoperoxidase stains, these cells stained positive for actin, myoglobin, and atrial natriuretic peptide, proving the cells are myocardial muscle cells. Electron microscopy showed numerous bundles of myofibrils with interspersed dense Z-bodies and pleomorphic mitochondria. Bromo-deoxyuridine incorporation confirmed that the cells were replicating their DNA. Thus, cell morphology, immunoperoxidase stains, electron microscopy, and cell proliferation testing showed these cells to be myocardiocytes undergoing DNA replication and mitosis. We must now reconsider our current thinking about myocardial development and investigate what factors contribute to the inhibition of myocardial cell proliferation after injury in vivo.

Bacteriological Techniques↗

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells?

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

Animals↗

Identification of seven putative origins of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA replication.

Seven putative origins of DNA replication (oris) were identified and located on the genome of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV), when an improved infection-dependent replication assay was used. A threefold higher yield of amplified plasmid was achieved when an m.o.i. of 1 was used (instead of 25), and another twofold increase was obtained when the interval between transfection and infection was extended from 5 to 24 h. Six of the putative oris were located in hr regions with homologous sequences. This suggests that all hrs in AcMNPV are bifunctional, i.e. have both ori and enhancer activity for transcription. In addition to the six hrs, the HindIII-K fragment of AcMNPV was also identified to carry a putative ori, although this fragment does not contain an hr region. However, the individual role of these seven oris during viral DNA replication, and whether they are all active simultaneously in vivo, is still unclear. The replication of an ori-containing plasmid starts at the same time (6 h post-infection) and proceeds at the same rate as viral DNA replication. A circular topology of ori-containing plasmids was a prerequisite for replication. Linear DNA, with an ori, did not replicate. Therefore, we suggest a theta structure or a rolling-circle as a model for baculovirus DNA replication.

Animals↗

Preferential loss of DNA polymerase alpha following suppression of replicative DNA synthesis of guinea pig macrophages by the immunostimulants muramyl dipeptide or lipopolysaccharide.

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate 3H-thymidine into trichloroacetic acid-insoluble fraction. In pulse-labeling experiments, the incorporated 3H-thymidine was detected in short fragments of DNA, which corresponded to the Okazaki fragments. These results indicate that the observed thymidine incorporation is due to nuclear DNA replication but not DNA repair. The observed DNA synthesis of the macrophages was remarkably suppressed when the cells were cultured in a presence of muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The significant decrease of DNA polymerase alpha activity was found in the cells treated with MDP or LPS. In contrast, the activity of polymerase beta was not at all affected by the same treatment.

Acetylmuramyl-Alanyl-Isoglutamine↗

Initiation of DNA replication in vitro by a DNA-membrane complex extracted from Bacillus subtilis.

Initiation of DNA replication has been observed in vitro with a DNA-membrane complex extracted from Bacillus subtilis. Antibiotics known to interfere with various aspects of initiation inhibited DNA synthesis significantly in vitro, whereas a mutant resistant to one inhibitor failed to respond to its presence. The inhibitory effects occurred primarily when the immediate RNA precursors (ribonucleoside triphosphates) were present in the assay solution but not significantly when the precursors were omitted. Complexes extracted from a temperature-sensitive initiation mutant were almost incapable of synthesizing DNA at the restrictive temperature but displayed extensive synthesis at the permissive temperature. A strong indication of semiconservative DNA synthesis was obtained in vitro after density-shift experiments involving incubation of the complex with a heavy-density DNA precursor, followed by neutral and alkaline CsCl density gradient centrifugation. A significant amount of chain elongation or repair (or both) was also observed.

Aminoglycosides↗

The spatial organization of sequences involved in initiation and termination of eukaryotic DNA replication.

Nuclear DNA is looped by attachment to a matrix or cage. As this cage is the site of DNA synthesis, sequences in the loops must attach before they are replicated. We have tested whether sequences which initiate replication are usually out in the loop and attach only during S phase or whether they are attached but quiescent during most of the cell-cycle. Sequences which permit plasmids to replicate autonomously in yeast cells (ARS's) are strong candidates for initiating sequences. Four different human ARS's all map remote from attachment points to the HeLa nuclear cage. In addition a potential terminus of replication is also remote from the cage. We conclude that sequences involved in initiation are usually out in the loop and that DNA synthesis is initiated by their attachment.

Base Sequence↗

The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication.

Human single-stranded DNA binding protein (human SSB) is a multisubunit protein containing polypeptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we identify the functions of the SSB and its individual subunits in SV40 DNA replication. The 70 kDa subunit was found to bind to single-stranded DNA, whereas the other subunits did not. Four monoclonal antibodies against human SSB were isolated which inhibited SV40 DNA replication in vitro. The antibodies have been designated alpha SSB70A, alpha SSB70B, alpha SSB70C, and alpha SSB34A to indicate which subunits are recognized. Immunolocalization experiments indicated that human SSB is a nuclear protein. Human SSB is required for the SV40 large tumor antigen-catalyzed unwinding of SV40 DNA and stimulates DNA polymerases (pol) alpha and delta. The DNA unwinding reaction and stimulation of pol delta were blocked by alpha SSB70C, whereas the stimulation of pol alpha by human SSB was unaffected by this antibody. Conversely, alpha SSB70A, -70B, and -34A inhibited the stimulation of pol alpha, but they had no effect on DNA unwinding and pol delta stimulation. None of the antibodies inhibited the binding of SSB to single-stranded DNA. These results suggest that DNA unwinding and stimulation of pol alpha and pol delta are required functions of human SSB in SV40 DNA replication. The human SSB 70-kDa subunit appears to be required for DNA unwinding and pol delta stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pol alpha.

Antibodies, Monoclonal↗

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Adenoviridae↗

Transcription factors and the control of DNA replication.

Initiation of DNA replication is mediated by the assembly of nucleoprotein complexes at cis-acting DNA sequences known as origins of replication. Recent studies in several systems show that accessory transcription factors accentuate origin utilization by multiple mechanisms. The remarkable similarities in the activities of accessory transcription factors at promoters and origins of replication suggest that transcription factors play a pivotal role in the regulation of chromosomal DNA synthesis in eukaryotic organisms.

Animals↗

Inhibition of simian virus 40 DNA replication in vitro by poly(ADP-ribosyl)ated diadenosine tetraphosphate.

Poly(ADP-ribosyl)ated diadenosine tetraphosphate was found to inhibit the in vitro replication of SV40 DNA. This inhibition was sensitive to preincubation of the polymer with either poly(ADP-ribose) glycohydrolase, diadenosine tetraphosphate (Ap4A):ADP phosphohydrolase, or an excess of free Ap4A. In contrast, the general catalytic activity of DNA polymerase was not inhibited by the poly(ADP-ribosyl)ated Ap4A when activated salmon sperm DNA was used as a template. These data suggest that inhibition of SV40 DNA replication by poly(ADP-ribosyl)ated Ap4A requires both the intact polymer and intact Ap4A moiety and is specific to events occurring during the initiation or elongation of a double-stranded template. Since both poly(ADP-ribose) and Ap4A accumulate in cultured mammalian cells following stresses which are accompanied by DNA strand breaks, these data are consistent with a model in which poly(ADP-ribosyl)ated Ap4A inhibits DNA replication following DNA damage.

Adenine Nucleotides↗

Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures.

Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.

Animals↗

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

Antigens, Polyomavirus Transforming↗

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5.

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.

Bacillus subtilis↗

DNA replication licensing factor.

DNA Replication Licensing Factor (RLF) is an essential activity required to restrict the duplication of genomic DNA to precisely once per cell cycle. Recent fractionation of RLF activity from Xenopus egg extracts has resulted in the identification of two essential components, RLF-B and RLF-M. RLF-M has been purified to homogeneity and has been shown to consist of a complex of proteins in the MCM/P1 family. RLF-B is still unidentified, but possible candidates for this activity have been identified in yeast. Elucidation of the RLF mechanism will provide important insights into the way that chromosome replication is controlled.

Animals↗

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗

Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking.

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.

Ascorbic Acid↗

Genomic specification and epigenetic regulation of eukaryotic DNA replication origins.

Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.

AT Rich Sequence↗

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.

Antimetabolites↗