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PubMed1991

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins. Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication. Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process. Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis. A possible role of the maturation promoting factor (MPF) is suggested.

Animals↗
PubMed1987

Cyclin (PCNA, auxiliary protein of DNA polymerase delta) is a central component of the pathway(s) leading to DNA replication and cell division.

Cyclin, also known as PCNA or the auxiliary protein of mammalian DNA polymerase delta, is a stable cell cycle regulated (synthesized mainly in S-phase) nuclear protein of apparent Mr 36,000 whose rate of synthesis correlates directly with the proliferative state of normal cultured cells and tissues. Cyclin (PCNA) is absent or present in very low amounts in normal non-dividing cells and tissues, but it is synthesized in variable amounts by proliferating cells of both normal and transformed origin. All available information indicates that this ubiquitous and tightly regulated DNA replication protein is a central component of the pathway(s) leading to DNA replication and cell division.

Animals↗
PubMed1986

Yeast DNA replication.

We have explored various strategies for exploiting the yeast genetic and biochemical system for understanding DNA replication. Because of the long time that has intervened between the isolation of random replication mutants of yeast and the identification of the gene products affected, an alternative approach to elucidating the molecular basis of replication has been needed. One such alternative involves purifying replication proteins, isolating the genes encoding them, and constructing the appropriate mutant from the cloned gene. We have applied this "reverse genetics" strategy to three different replication activities: DNA polymerases, single-stranded DNA binding proteins, and proteins that bind to autonomously replicating sequence (ARS) elements.

DNA Polymerase I↗
PubMed1983

Structure and function of DNA binding proteins from revertants of adenovirus type 5 mutants with a temperature-sensitive DNA replication.

H5ts107 and H5ts125 are two adenoviruses type 5 (Ad5) mutants with a temperature-sensitive DNA replication. Both mutants contain an altered gene encoding the DNA binding protein (DBP). We have established by nucleotide sequence analysis that both mutants carry exactly the same mutation in the DBP gene resulting in the substitution of a proline residue at position 413 in the wild-type DBP amino acid sequence (529 amino acid residues long) by a serine residue. Revertants of H5ts107 and H5ts125, which are temperature independent in plaque efficiency and growth in HeLa cells at 32 degrees and 39 degrees, were characterized by nucleotide sequence analysis of their DBP genes. Four types of revertants could be distinguished: revertants with the wild-type DBP amino acid sequence (type I) and, revertants carrying, in addition to the original H5ts107/H5ts125 mutation at position 413, intragenic second site mutations at position 508 histidine leads to tyrosine (type II), at position 352 glycine leads to aspartic acid (type III), and at position 347 alanine leads to proline (type IV), respectively. All intragenic second site mutations are located, together with the H5ts107/H5ts125 mutation, in the C-terminal 45-kD fragment of the adenovirus DBP molecule. This provides further evidence that this part of the DBP molecule plays an important role in viral DNA replication. Phenotypic characterization of the revertants (J.C. Nicolas, F. Suarez, A.J. Levine, and M. Girard (1981), Virology 108, 521-524; (J.C. Nicolas, D. Ingrand, P. Sarnow, and A.J. Levine (1982), Virology 122, 481-485) has shown that the second site mutations reveal additional functional domains in the DBP molecule.

Adenoviruses, Human↗
PubMed1997

cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication.

The A genomic component of the geminivirus tomato golden mosaic virus (TGMV) contains a 5' intergenic sequence that includes the overlapping AL61 promoter and positive-strand origin of DNA replication. The TGMV AL1 protein negatively regulates its own transcription and mediates origin recognition by binding to a repeated motif shared by the AL61 promoter and the viral origin. We examined a series of truncated or mutated 5' intergenic regions in transient expression and replication assay to identify other DNA sequences that contribute to TGMV promoter and origin function. These experiments revealed that negative regulation of the AL61 promoter is complex, involving multiple cis-acting sequences and the AL1 and AL4 proteins, which acted through different DNA elements. We also found that mutation of the TATA box motif in the AL61 promoter reduced overall transcriptional activity and AL1-mediated repression, confirming the importance of this sequence in promoter function. Mutation of a G-box consensus sequence was highly detrimental to AL61 transcription and abolished AL1 sensitivity, suggesting that AL1 interferes with transcriptional activation. Cotransfection experiments showed that the TATA box and G-box motif mutations also impaired viral DNA replication in the presence of a wild-type origin but had no effect in its absence, demonstrating that these transcriptional motifs also function as replication efficiency elements.

Base Sequence↗
PubMed1980

Purification and properties of a DNA ligase from a soluble DNA replication complex.

A DNA ligase has been purified from a subnuclear soluble replication complex isolated from adenovirus type 2-infected human KB cells. DNA ligase activity could not be demonstrated using an exogenous template until the complex was dissociated, suggesting that the ligase activity may be a component of the complex. The purified enzyme was free of endonuclease, exonuclease, 5'-nucleotidase, and phosphatase activities, and had a molecular weight of 105 000, as estimated by sedimentation in a glycerol gradient. The ligase requires ATP and a divalent cation for activity. The optimum of the reaction is at pH 7.8 in 50--100 mM Tris-HCl buffer and 10--20 mM MgCl2. Monovalent salts greatly stimulate ligase activity and the optimum was found at 150 mM. The reaction is very sensitive to high temperature; maximum activity was observed at 25--30 degrees C. ATP is the sole required cofactor and NAD, dATP and GTP could not replace the requirement for ATP. The Km for ATP is 60 microM. The Km for DNA is 250 microgram/ml or 1.6 nmol of terminal phosphate/ml and thus the enzyme shows relatively weak affinity for exogenous DNA. The maximum conversion of 32P into a phosphatase-resistant form is approximately 1.3% of the total, whereas T4 ligase, under the same conditions, can convert more than 25% of phosphate into a resistant form.

Adenoviruses, Human↗
PubMed1994

A developmental checkpoint couples the initiation of sporulation to DNA replication in Bacillus subtilis.

Spore formation in Bacillus subtilis requires the generation of two distinct cell types, each with an active chromosome that becomes committed to a defined program of gene expression. Here we show that a developmental checkpoint couples the initiation of sporulation, and the subsequent formation of these two cell types, to DNA replication early during development. Inhibiting the initiation of chromosomal replication prevents the onset of sporulation and inhibits expression of several genes that are normally induced early during development. This defect in gene expression is due to inhibition of the multi-component phosphorylation pathway needed to activate the developmental transcription factor encoded by spo0A. The target affected by inhibiting the initiation of replication is neither Spo0A nor the major kinase, KinA, needed for production of Spo0A approximately P. Rather, the target appears to be one of the proteins that transfers phosphate from the kinase to the Spo0A transcription factor. The signal that couples activity of the phosphorelay to the initiation of DNA replication is different from the previously described DNA damage signal that inhibits the phosphorelay during SOS induction in a recA-dependent response. Thus, DNA replication as well as DNA damage signals control production of Spo0A approximately P and initiation of sporulation.

Bacillus subtilis↗
PubMed1990

Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase.

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.

Burkitt Lymphoma↗
PubMed2004

DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior.

The DNA replication checkpoint maintains replication fork integrity and prevents chromosome segregation during replication stresses. Mec1 and Rad53 (human ATM/ATR- and Chk2-like kinases, respectively) are critical effectors of this pathway in yeast. When treated with replication inhibitors, checkpoint-deficient mec1 or rad53 mutant fails to maintain replication fork integrity and proceeds to partition unreplicated chromosomes. We show that this unnatural chromosome segregation requires neither the onset of mitosis nor APC activation, cohesin cleavage, or biorientation of kinetochores. Instead, the checkpoint deficiency leads to deregulation of microtubule-associated proteins Cin8 and Stu2, which, in the absence of both chromosome cohesion and bipolar attachment of kinetochores to microtubules, induce untimely spindle elongation, causing premature chromosome separation. The checkpoint's ability to prevent nuclear division is abolished by combined deficiency of microtubule-destabilizing motor Kip3 and Mad2 functions. Thus, the DNA replication checkpoint prevents precocious chromosome segregation, not by inhibiting entry into mitosis as widely believed, but by directly regulating spindle dynamics.

Blotting, Northern↗
PubMed1993

The C-terminal third of UL42, a HSV-1 DNA replication protein, is dispensable for viral growth.

UL42 is the herpes simplex virus type 1 DNA polymerase (Pol) accessory protein and is required for viral DNA replication and growth. Previous results from this laboratory demonstrated that the N-terminal two thirds of the protein contains all of the biochemical activities of the protein which can be measured in vitro. These activities include dsDNA-binding, association with DNA polymerase, and stimulation of polymerase activity. To better understand the functions of UL42 in infected cells, we have isolated and characterized two viral recombinants, UL42lacZ and n338. In the mutant virus UL42lacZ, the UL42 gene was disrupted by insertion of the Escherichia coli lacZ gene, while in the mutant virus n338, a termination codon was introduced after amino acid position 338. Analysis of the mutant phenotypes suggest that (1) the first 338 residues of UL42 retain all the functions necessary for viral DNA replication and growth in lytic infection, (2) localization of UL42 to the cell nucleus is independent of Pol, and (3) localization of ICP8 (ssDNA-binding protein) to prereplication sites is independent of functional UL42.

Animals↗
PubMed1988

Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.

Animals↗
PubMed2004

Protein and sequence requirements for the recruitment of the human origin recognition complex to the latent cycle origin of DNA replication of Epstein-Barr virus oriP.

Initiation of DNA replication from within the Epstein-Barr virus (EBV) latent cycle origin oriP occurs once per cell cycle and is almost entirely dependent upon cellular proteins. The human origin recognition complex (ORC) is recruited to oriP and orchestrates the events that lead to the initiation of replication. EBNA-1, the sole viral protein required for oriP-plasmid replication, binds four sites within the replicator but the role(s) it plays in the replication of oriP plasmids has not been elucidated. We investigated the recruitment of ORC to oriP in vivo and show that the binding of EBNA-1 to the replicator is necessary for the association of the ORC subunit Orc2 with the replicator. The minimal replicator of oriP consists of two EBNA-1 binding sites flanked by perfect 14-bp inverted repeats (a and b), but these repeats are dispensable for the association of Orc2 with the replicator. A mutational analysis of the 14-bp repeats provided additional support for a role for the telomere repeat binding protein 2 in oriP replicator function. We show that nucleotide differences between the oriP replicator of the B95-8 and Raji EBV genomes are not solely responsible for the inefficient utilization of this origin in the Raji EBV genome.

Binding Sites↗
PubMed1984

In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells.

DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM. The initial incorporation rate at 10 microM TTP concentration was about 12% of the in vivo synthesis and declined very strongly within 1 h. A similar decline of the incorporation rate was found at 0.12 microM TTP concentration. DNAase I digestion of DNA-matrix complexes obtained from isolated nuclei in 2 M NaCl revealed that newly replicated DNA was preferentially bound to the nuclear matrix. A similar digestion with S1 nuclease caused a selective release of short duplexes of Okazaki fragments with the complementary parental strand. The results show that in vivo replication continues in permeabilized cells in an almost unchanged way, except for a gradual decline of its rate which is mainly due to inactivation of one or more essential components.

Animals↗
PubMed1985

Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression.

We constructed and analyzed a series of deletion mutants in the noncoding regulatory region of tsa polyomavirus DNA to identify some of the sequences critical to the DNA replication origin and to the expression of the viral early genes in vivo. By using both transient and long-term assays under conditions where the influence of large T antigen (T-Ag) in replication or autoregulation was minimized, we observed no more than a 30% reduction in early gene expression upon removal of the CAAT or TATA elements or both. These assays demonstrated a predominant effect of upstream promoter or enhancer elements and indicated that removal of the CAAT or TATA boxes did not significantly affect viral early gene expression. Studies on the replicative ability of these mutants in mouse cells constitutively expressing the polyoma early proteins revealed that the removal of DNA sequences contained within a previously identified T-Ag high-affinity binding site (nucleotides 39 to 64) abolished viral DNA replication, whereas removal of two other high-affinity sites, closer to the early mRNA cap sites, did not. Furthermore, a deletion including this same high-affinity site plus a low-affinity binding site within the 32-base-pair palindrome of the origin core sequences eliminated the ability of the viral large T-Ag to efficiently repress early gene transcription. It is thus possible that the origin-proximal high-affinity T-Ag binding site is involved in both of the functions of large T-Ag, i.e., the initiation of viral DNA replication and the autoregulation of early gene transcription.

Animals↗
PubMed1992

Mechanism of protein priming DNA replication of B.subtilis phage M2.

B.subtilis phage M2 uses a protein, instead of RNA, as the primer of its DNA replication. Hence this protein encoded in the phage genome is called as the primer protein (PP). At the initiation of DNA replication, a hetero dimer complex with its own DNA polymerase and the PP supposed to interact with the terminal protein (TP), which is covalently bound to the template DNA (TP-DNA). PP contained an important adhesive amino acid sequence, Arg-Gly-Asp (RGD), near the carboxyl terminal. We have recently showed that the synthetic RGD peptide inhibited the transfection of phage M2. By site-directed mutagenesis, we introduced different amino acid into the RGD site of PP. These altered PP decreased obviously the priming activity in vitro.

Bacillus Phages↗
PubMed2001

DNA replication efficiency depends on transcription factor-binding sites.

Naturally arising variants of simian virus 40 (SV40), generated by serial passage of the virus at high multiplicities of infection, provide important insight into the role of transcription factor-binding sites in enhancing DNA replication. Although the variants that arise from numerous recombination events are the result of selective pressure to replicate more efficiently than the other variants in the infection, there is no transcription pressure. Therefore, it is interesting that a minimum of two viral Sp1 transcription factor-binding sites are retained and that host AP-1 and NF-1 transcription factor-binding sites are incorporated into the 100-bp regulatory region that maximizes DNA replication in these variants. We cotransfected COS-1 cells (that provide viral large T antigen for DNA replication) to examine the effect of transcription factor-binding sites on the replication of plasmid constructs that contain the SV40 origin of replication (ori). The level of relative replication efficiency (RRE) depends on the number and type of transcription factor-binding sites. Replication increases as the number of transcription factor-binding sites increases within the regulatory region of the variants; AP-1 sites are more effective than NF-1 transcription factor-binding sites. Competition between constructs in transfections magnifies the difference in their RREs. The results indicate that transcription factor-binding sites play an important role in enhancing DNA replication.

Animals↗
PubMed1993

Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication.

The UL30 protein of herpes simplex virus type 1 (HSV-1) is a catalytically active DNA polymerase which is present in virus infected cells in a heterodimeric complex with an accessory subunit, the UL42 polypeptide. Both proteins are essential for viral DNA synthesis but because the UL42 protein is much more abundant it has been difficult to determine whether its role is related to, or independent of, its interaction with the UL30 protein in vivo. Since the C-terminal region of UL30 has been shown to be important for interaction with the UL42 protein but dispensable for DNA polymerase activity, a recombinant baculovirus which overexpresses a UL30 protein truncated by 27 amino acids at its C-terminus was constructed and used to assess the significance of the protein-protein interaction. The mutated protein was as active as wildtype (wt) UL30 in a DNA polymerase assay in which activated calf thymus DNA was used as template. However, in contrast to the wt protein, the activity of the truncated polymerase on this template was not stimulated by addition of purified UL42. A monoclonal antibody against the UL42 protein co-precipitated the full length but not truncated polymerase from extracts of cells which had been co-infected with a UL42-expressing recombinant baculovirus. Finally, the truncated protein was not active in a transient assay for HSV-1 origin-dependent DNA replication performed in insect cells in tissue culture. These results indicate that sequences at the C-terminus of the UL30 protein which are dispensable for DNA polymerase activity play essential roles both in viral DNA replication and interaction with the UL42 protein, and strongly suggest that the interaction between the proteins is important in vivo.

Amino Acid Sequence↗
PubMed1983

Initiation of simian virus 40 DNA replication in vitro.

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.

Antigens, Viral, Tumor↗