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PubMed2004

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells.

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.

Animals↗
PubMed2003

Positive- and negative-acting signals combine to determine differential RNA replication from the paramyxovirus simian virus 5 genomic and antigenomic promoters.

The cis-acting signals found at the 3' ends of the genomic and antigenomic RNAs are a major factor determining the level of paramyxovirus RNA replication from each promoter. Using a minigenome system that reconstitutes SV5 RNA synthesis from cDNA-derived components, we show here that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication approximately 14-fold lower than that seen from the antigenomic promoter (AGP). The goal of this study was to identify cis-acting signals responsible for differential levels of RNA replication from the SV5 GP and AGP. We have previously shown that the SV5 AGP contains three sequence-dependent elements (CRI, CRII, and Region III) that are separated by sequence-independent spacer regions. Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. Minigenomes containing a substitution of the AGP CRI, CRII, or Region III elements alone in place of the corresponding GP sequences did not show enhanced levels of RNA replication. However, transfer of both the AGP 3' terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to approximately 40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20-50) located between CRI and Region III. Importantly, transfer of nonviral sequences in place of GP bases 20-50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. These data indicate that differential levels of RNA replication from the SV5 GP and AGP are due to a combination of positive-acting signals in the AGP (CRI and Region III) and a negative-acting signal in the GP (bases 20-50). Possible functions for the SV5 promoter elements in determining RNA replication levels are proposed.

Amino Acid Sequence↗
PubMed1993

O6-methylguanine in DNA inhibits replication in vitro by human cell extracts.

To study the effects of methylation damage on DNA replication in vitro, the plasmid pSVori containing the SV40 origin of replication was reacted with N-methyl-N-nitrosourea and used as a substrate for SV40 T antigen dependent replication by HeLa cell extracts. The plasmid was methylated with a range of N-methyl-N-nitrosourea concentrations that introduced an average of 0.3-2.5 O6-methylguanine and equal amounts of 3-methyladenine lesions per DNA molecule. When methylated plasmid was incubated with extract of Mex-HeLaMR cells under conditions favoring DNA replication, an impairment of replication was observed as the accumulation of incompletely replicated form II plasmid molecules. These extracts simultaneously performed a T antigen independent, DpnI-sensitive DNA repair synthesis that increased with increasing DNA damage. Subtraction of this repair DNA synthesis revealed that methylation inhibited overall replication. At low levels of methylation (< or = 1 O6-methylguanine and < or = 1 3-methyladenine lesion per plasmid), inhibition was transient, while more extensive damage resulted in apparently irreversible inhibition of replication. Removal of O6-methylguanine by pretreatment of the methylated plasmid with purified human O6-methylguanine-DNA methyltransferase restored replication to almost normal levels. When the methylated plasmid was replicated by extracts of Mex+ HeLaS3 cells proficient in the repair of O6-methylguanine, a lower level of inhibition and less repair DNA synthesis was observed. The inhibition of DNA synthesis and the stimulation of repair DNA synthesis are thus both largely due to the presence of O6-methylguanine in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Polyomavirus Transforming↗
PubMed1991

Gene replication in the presence of aphidicolin.

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.

Animals↗
PubMed1987

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication.

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Factors influencing the replication of somatic coliphages in the water environment.

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.

Bacteroides fragilis↗
PubMedDate not supplied

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression.

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.

Adenoviridae↗
PubMed1994

Chemical self-replication of palindromic duplex DNA.

Molecular replication, a fundamental process of life, has in recent years been the subject of laboratory investigations using simple chemical systems. Whereas the work of Rebek's group has focused on molecular architectures not known in living systems, self-replicating and template-based self-assembling systems based on nucleotides are regarded as potential models for exploring the evolution of replicating systems on the early Earth. Previous replicating oligonucleotides have been of the single-stranded, self-complementary type: small oligonucleotide fragments are assembled on a pre-existing template and linked to form an exact copy of the template. This process cannot easily be reiterated, however, because of the strong binding of the newly formed strand to the original template. Furthermore, DNA replication in living systems operates by complementarity rather than self-complementarity--each newly assembled strand is complementary to, rather than identical to, its template--and the replication process starts and finishes with double helices. Here we report the self-replication of palindromic (symmetrical) duplex DNA-like oligonucleotides, 24 monomers long, in the absence of enzymes by means of a cycle that transfers information from template to copy and is potentially capable of extension to include non-symmetrical sequences, selection and mutation. Replication proceeds by a chemical process involving the formation of an intermediate triplex structure, and is sequence-selective in the sense that mismatches impair its efficiency. These results indicate that DNA-like double-helical molecules can replicate without assistance from proteins, a finding that may be relevant both to the appearance of replicating systems on the early Earth and to the development of new approaches to DNA amplification.

Base Sequence↗
PubMed1995

MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells.

An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis. We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein. Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.

Amino Acid Sequence↗
PubMed2003

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Animals↗
PubMed2006

Selective replication of E1B55K-deleted adenoviruses depends on enhanced E1A expression in cancer cells.

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

Adenoviridae↗
PubMed2004

The chromatin remodeling complex NoRC controls replication timing of rRNA genes.

The ATP-dependent chromatin remodeling complex NoRC silences a fraction of mammalian ribosomal RNA genes (rDNA) by establishing heterochromatic structures at the rDNA promoter. Here we show that NoRC also plays a role in replication timing of rDNA. rDNA is replicated in a biphasic manner, active genes ( approximately 60%) replicating early and silent ones ( approximately 40%) replicating late in S-phase. The chromatin structure that marks active and silent rDNA repeats is propagated during cell division. To examine the function of NoRC in epigenetic inheritance and replication timing, we have monitored the chromatin structure, transcriptional activity and replication timing of rDNA in a cell line that moderately overexpresses NoRC. NoRC is exclusively associated with late-replicating rDNA arrays. Overexpression of NoRC silences rDNA transcription, reduces the size and number of nucleoli, impairs cell proliferation and resets replication timing from early to late. The results demonstrate that NoRC is an important determinant of replication timing and epigenetic marks are heritably maintained through DNA replication.

Adenosine Triphosphate↗
PubMed2004

Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes.

Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited. We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes. In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome. This finding contrasts with the coordinated timing of both ends of each chromosome in yeast. Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later. Finally, replication timing of some but not all telomeres was influenced by telomere length. Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action. The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci.

Animals↗
PubMed2001

Topological challenges to DNA replication: conformations at the fork.

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (-) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (-) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.

DNA Replication↗
PubMed2001

Rescue of arrested replication forks by homologous recombination.

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

DNA Replication↗
PubMed2003

Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation.

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

Antiviral Agents↗
PubMed1977

Terminus region of the chromosome in Escherichia coli inhibits replication forks.

Induction of prophage P2sig5 at 42 degrees caused replication of the bacterial chromosome in a dnaA mutant of Escherichia coli. The P2sig5 is integrated in this strain near the metG locus, which is at min 47 on the genetic map. The regions of the chromosome replicated after prophage induction have been determined by means of DNA-DNA hybridization with various DNAs obtained from Proteus mirabilis/E. coli F' merogenotes and from lambda specialized transducing phage. The replication was initiated at the prophage site and was bidirectional. Most of the replication occurred in a counterclockwise direction on the genetic map, and the replication quickly proceeded to the aroD locus (min 37). The replication forks were retarded between aroD and rac (min 31) loci, although the rac locus was finally replicated. A more severe inhibition of replication occurred between the rac and trp (min 27) loci. It is proposed that the replication terminus is near the rac locus and that the terminus inhibits replication forks.

Chromosomes, Bacterial↗
PubMed1988

CDC7-dependent protein kinase activity in yeast replicative-complex preparations.

A protein kinase activity was identified in preparations of DNA-replicative complex from the budding yeast Saccharomyces cerevisiae. The activity phosphorylated only a few of the endogenous proteins in the replicative fraction, and it displayed a marked preference for a 48-kDa polypeptide. Despite this relative specificity, the protein kinase activity was capable of utilizing exogenously added histone as substrate. The 48-kDa polypeptide was phosphorylated on serine residue(s) exclusively by the endogenous activity in the replicative-complex preparation. The activity was not stimulated by cAMP, cGMP, Ca2+/phosphatidylserine/diacylglycerol, or Ca2+/calmodulin. It did not utilize Ca2+ or Zn2+ in the place of Mg2+, and Mn2+ was only 22% as effective in fulfilling the divalent-cation requirement. Most importantly, the protein kinase activity was heat-sensitive in replicative fractions from the cell division cycle 7 (cdc7) mutant, which arrests at or close to the G1/S boundary of the cell cycle at restrictive temperature. Thus, the activity is CDC7-dependent. An effect of heat treatment on replicating activity in the replicative fraction from cdc7 cells was also found. This result and the finding that the protein kinase activity copurified with replicating activity in the preparations suggest that the CDC7 gene product and the protein kinase activity, whether or not they are the same entity, interact with yeast replicative complex. All of these results raise the possibility that phosphorylation of components of the replication machinery may play a role in the control of initiation of DNA replication during the cell cycle. It is possible that the phosphorylation observed is part of a protein kinase cascade that regulates progress through the G1 phase of the cell cycle.

DNA Replication↗