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PubMed2002

Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.

Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

Animals↗

PubMed2006

Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression.

Origins of DNA replication are licensed in G1 by recruiting the minichromosome maintenance (MCM) proteins to form a prereplicative complex (pre-RC). Prior to initiation of DNA synthesis from each origin, a preinitiation complex (pre-IC) containing Cdc45 and other proteins is formed. We report that Cdc7-Dbf4 protein kinase (DDK) promotes assembly of a stable Cdc45-MCM complex exclusively on chromatin in S phase. In this complex, Mcm4 is hyperphosphorylated. Studies in vitro using purified DDK and Mcm4 demonstrate that hyperphosphorylation occurs at the Mcm4 N terminus. However, the DDK substrate specificity is conferred by an adjacent DDK-docking domain (DDD), sufficient for facilitating efficient phosphorylation of artificial phosphoacceptors in cis. Genetic evidence suggests that phosphorylation of Mcm4 by DDK is important for timely S phase progression and for cell viability upon overproduction of Cdc45. We suggest that DDK docks on and phosphorylates MCM proteins at licensed origins to promote proper assembly of pre-IC.

Amino Acid Motifs↗

PubMed1995

Examination of conditions affecting the efficiency of HVS-1 amplicon packaging.

Defective herpes simplex virus type 1 vectors (HSV amplicons) have been used as vehicles for efficiently delivering foreign genes into non-dividing cells such as neutrons in vitro and in vivo. This system is useful for studying neuronal physiology and may have potential for human gene therapy of neuronal disorders. The preparation of infectious amplicon particles is normally achieved by transfecting amplicon plasmid DNA, which contains the HSV replication origin and packaging signal, into mammalian cell lines followed by infection of the cells with HSV helper virus. This allows for replication and packaging of both viral and amplicon plasmid DNA. To improve the packaging efficiency of amplicons, several parameters involved in the packaging process were investigated. By introducing the SV40 DNA replication origin into an amplicon plasmid and prereplicating it before HSV infection, it was demonstrated that the existing amount of amplicon DNA prior to infection in the cells is not a rate-limiting step during HSV packaging. In addition, it was shown that the yield of the packaged amplicon particles can be improved by: (1) using a relatively small amount of HSV helper virus up to multiplicity of infection (m.o.i.) equal to 0.1 at infection; (2) infecting with HSV helper virus at 2 or 3 days post-transfection; and (3) passaging the initial packaged amplicon stocks 1-2 times on fresh host cells.

Animals↗

PubMed1997

Role of TrfA and DnaA proteins in origin opening during initiation of DNA replication of the broad host range plasmid RK2.

The Escherichia coli protein DnaA and the plasmid RK2-encoded TrfA protein are required for initiation of replication of the broad host range plasmid RK2. The TrfA protein has been shown to bind to five 17-base pair repeat sequences, referred to as iterons, at the minimal replication origin (oriV). Using DNase I footprinting and a gel mobility shift assay, purified DnaA protein was found to bind to four DnaA consensus binding sequences immediately upstream of the five iterons at the RK2 origin of replication. Binding of the TrfA protein to the iterons results in localized strand opening within the A+T-rich region of the replication origin as determined by reactivity of the top and bottom strands to potassium permanganate (KMnO4). The presence of either the E. coli DnaA or HU protein is required for the TrfA-mediated strand opening. Although the DnaA protein itself did not produce an RK2 open complex, it did enhance and/or stabilize the TrfA-induced strand opening.

Adenosine Triphosphate↗

PubMed2001

Assembly of the human origin recognition complex.

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.

DNA-Binding Proteins↗

PubMed2002

Human kin17 protein directly interacts with the simian virus 40 large T antigen and inhibits DNA replication.

Kin17 is an evolutionarily conserved DNA-binding protein, which forms intranuclear foci in proliferating cells. Recent data have suggested that human kin17 protein is associated with cell proliferation and unrepaired DNA lesions. Herein, we show that human fibroblasts (MRC5-V2 and CHSV4) immortalized with SV40 overexpress endogenous kin17 protein, as compared with normal diploid human fibroblasts. We observed that certain carcinoma cell lines also up-regulated kin17 protein, suggesting that increased kin17 protein levels may be a consequence of the immortalized phenotype. We report here that the endogenous kin17 protein is located in nucleoplasmic foci and colocalizes with SV40 large T antigen. Purification of human kin17 protein allowed analysis of the physical interaction with T antigen by several in vitro and in vivo assays. Large T antigen and human kin17 protein are part of the same high molecular weight multiprotein complex in human cells. Furthermore, human kin17 protein interacts with T antigen bound to the SV40 DNA origin of replication. Strikingly, the overexpression of human kin17 protein in vivo and the introduction of increased amounts of human kin17 protein in an in vitro assay reduced T-antigen-dependent DNA replication, suggesting that kin17 protein may be involved in the DNA replication process in human cells.

Animals↗

PubMed1977

Origin of replication of pBR345 plasmid DNA.

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.

Base Sequence↗

PubMed2004

Requirement of a 12-base-pair TATT-containing sequence and viral lytic DNA replication in activation of the Kaposi's sarcoma-associated herpesvirus K8.1 late promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by trans.

Base Sequence↗

PubMed2004

Density matters: the semiconservative replication of DNA.

The semiconservative mode of DNA replication was originally documented through the classic density labeling experiments of Matthew Meselson and Franklin W. Stahl, as communicated to PNAS by Max Delbruck in May 1958. The ultimate value of their novel approach has extended far beyond the initial implications from that elegant study, through more than four decades of research on DNA replication, recombination, and repair. I provide here a short historical commentary and then an account of some developments in the field of DNA replication, which closely followed the Meselson-Stahl experiment. These developments include the application of density labeling to discover the repair replication of damaged DNA, a "nonconservative" mode of synthesis in which faulty sections of DNA are replaced.

Biophysics↗

PubMedDate not supplied

Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing.

Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells. To achieve this coordinated replication, eukaryotic cells employ an ordered series of steps to form several key protein assemblies at origins of replication. Major progress has recently been made in identifying the enzymes, and other proteins, of DNA replication that are recruited to origin sites and the order in which they are recruited during the process of replication. More than 20 proteins, including DNA polymerases, have been identified as essential components that must be preassembled at replication origins for the initiation of DNA synthesis. Of the polymerases, DNA polymerase alpha-primase (pol alpha) is of particular importance since its function is fundamental to understanding the initiation mechanism of eukaryotic DNA replication. DNA must be replicated with high fidelity to ensure the accurate transfer of genetic information to progeny cells, and decreases in DNA pol alpha activity and fidelity, which are coordinated with cell cycle progression, have been shown to be important facets of a probable intrinsic cause of genetic alterations during in vitro ageing. This has led to the proposal that pol alpha activity and function is one of the crucial determinants in ageing. In this review we summarize the current state of knowledge of DNA pol alpha function in the regulation of DNA replication and focus in particular on its interactive tasks with other proteins during in vitro ageing.

Cell Cycle↗

PubMed1989

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation F101, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1:67:21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.

Animals↗

PubMed1991

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

Antibodies, Monoclonal↗

PubMed1996

Initiation of baculovirus DNA replication: early promoter regions can function as infection-dependent replicating sequences in a plasmid-based replication assay.

From the results of transient plasmid-based replication assays, it has been postulated that homologous regions (hrs) of Autographa californica nuclear polyhedrosis virus (AcMNPV) function as origins of viral DNA replication. However, these assays vary in specificity according to the methodology used and may not be dependent solely on the presence of hr sequences. To determine the role that hrs and other sequences might play in the replication process, a series of plasmids containing specific deletions of various hrs was generated and tested in a standardized replication assay. Deletion of the AcMNPV hr2 and hr5 sequences abolished the ability of plasmids to replicate in the standard infection-dependent replication assay, while deletion of hr1, hr3, and hr4a sequences decreased but did not eliminate plasmid replication in this assay. Plasmids carrying the complete ie-2 and pe38 genes, the ie-1 gene upstream region, or a variety of baculovirus genes including 11 early promoter regions were also able to replicate in virus-infected cells, suggesting that early viral promoter sequences could also function as putative origins of replication. These data suggest that the standard infection-dependent replication assay may identify a broad range of infection-dependent replicating sequences, only one or a few of which may represent genuine viral origins used by the virus in vivo. We propose a model suggesting that the selection of replication initiation sites may be imposed directly by chromatin structure and indirectly by primary sequence and that the process of viral DNA replication may be linked with viral transcription.

Baculoviridae↗

PubMed2002

The mcm5-bob1 bypass of Cdc7p/Dbf4p in DNA replication depends on both Cdk1-independent and Cdk1-dependent steps in Saccharomyces cerevisiae.

The roles in DNA replication of two distinct protein kinases, Cdc7p/Dbf4p and Cdk1p/Clb (B-type cyclin), were studied. This was accomplished through a genetic and molecular analysis of the mechanism by which the mcm5-bob1 mutation bypasses the function of the Cdc7p/Dbf4p kinase. Genetic experiments revealed that loss of either Clb5p or Clb2p cyclins suppresses the mcm5-bob1 mutation and prevents bypass. These two cyclins have distinct roles in bypass and presumably in DNA replication as overexpression of one could not complement the loss of the other. Furthermore, the ectopic expression of CLB2 in G1 phase cannot substitute for CLB5 function in bypass of Cdc7p/Dbf4p by mcm5-bob1. Molecular experiments revealed that the mcm5-bob1 mutation allows for constitutive loading of Cdc45p at early origins in arrested G1 phase cells when both kinases are inactive. A model is proposed in which the Mcm5-bob1 protein assumes a unique molecular conformation without prior action by either kinase. This conformation allows for stable binding of Cdc45p to the origin. However, DNA replication still cannot occur without the combined action of Cdk1p/Clb5p and Cdk1p/Clb2p. Thus Cdc7p and Cdk1p kinases catalyze the initiation of DNA replication at several distinct steps, of which only a subset is bypassed by the mcm5-bob1 mutation.

CDC2 Protein Kinase↗

PubMed2000

Human p32: a coactivator for Epstein-Barr virus nuclear antigen-1-mediated transcriptional activation and possible role in viral latent cycle DNA replication.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is required for the maintenance of the viral chromosome in latently infected, proliferating cells and plays a role in latent cycle DNA replication. EBNA-1 also functions as a positive and negative regulator of EBV gene expression. We have investigated the interaction of EBNA-1 with p32, a host mitochondrial protein that associates with EBNA-1 in EBV-positive Burkitt's lymphoma cells. Using a chromatin immunoprecipitation assay, we found that a fraction of p32 localizes to the viral latent cycle origin of DNA replication oriP in vivo. p32 binds EBNA-1 independently of other proteins or DNA. EBNA-1 variants lacking one of two p32 binding elements did not interact stably with p32 in cultured cells and were defective for both transcriptional activation of a reporter gene linked to oriP FR and replication and/or maintenance of a plasmid bearing oriP. These results support a role for p32 in transcriptional activation by EBNA-1 and suggest that p32 plays a role in EBV latent cycle DNA replication.

Animals↗

PubMed1990

Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.

Adenosine Triphosphatases↗

PubMed2006

PCNA is a cofactor for Cdt1 degradation by CUL4/DDB1-mediated N-terminal ubiquitination.

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Cell Cycle Proteins↗

PubMed2001

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum.

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.

Animals↗