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At least 667 records · Page 37Linked to original sources
PubMed1996

Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases.

An important aspect of cell behaviour is that DNA replication happens only once per cell cycle. Replicated DNA is unable to re-replicate until cell division has occurred. Unreplicated DNA is in a replication-competent or 'licensed' state. The ability to replicate is lost in S phase and regained following passage through mitosis. Recent evidence has implicated an MCM (minichromosome maintenance) protein complex and the Cdc6 protein in determining replication competence. Regeneration of replication competence upon passage through mitosis entails changes in protein kinase activity, of which the MCMs are a likely target. Features of the mechanism that restricts DNA replication to once per cell cycle appear to be conserved throughout eukaryotes.

Journal Article↗
PubMed2005

Molecular dynamics of a food carcinogen-DNA adduct in a replicative DNA polymerase suggest hindered nucleotide incorporation and extension.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary PhIP is G-->T transversions. The major PhIP-derived DNA adduct is to C8 of guanine. We have investigated this adduct in a PhIP-induced mutational hotspot 5'-GGGA-3' of the Apc tumor suppressor gene, frequently mutated in mammalian colon tumors. We have carried out a molecular dynamics study to elucidate on a structural level nucleotide incorporation and extension opposite this major adduct during replication. The PhIP adduct was modeled into the ternary complex closed conformation of DNA polymerase RB69, at incorporation and extension positions, with normal cytosine or mismatched partner adenine. RB69 polymerase is a member of the B family as are most replicative eukaryotic DNA polymerases such as DNA polymerase alpha. These systems were subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite an incoming dCTP or dATP. In the case of the normal partner, disturbance to the active site is observed at the incorporation step, but there is less perturbance in the extension simulation. In the case of the mismatched partner, a less disturbed active site is observed during the incorporation step, but extension appears to be more difficult. Disturbances include adverse impacts on Watson-Crick hydrogen bonding in the nascent base pair, on the distance between the alpha-phosphate of the incoming dNTP and the primer terminus 3'-OH, and on critical protein interactions with the dNTP. However, in all of these cases, a near reaction ready distance (within 3.5 angstroms) between the 3'-terminal oxygen of the primer and the Palpha of the incoming nucleotide triphosphate is sampled occasionally (0.4-23.5% of the time). Thus, error-free bypass or the induction of a G-->T transversion mutation could occur at times and contribute to an extent to the mutagenic effect of PhIP. Polymerase stalling would be the more common outcome and in vivo could lead to switch to an error-prone bypass polymerase.

Amino Acids↗
PubMed2006

The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication.

Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases alpha and epsilon onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase alpha. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

Amino Acid Sequence↗
PubMed1987

A comparative study of hepatic DNA repair, DNA replication and hepatotoxicity in the CD-1 mouse following multiple administrations of dimethylnitrosamine.

The objective of this study was to quantify hepatic DNA repair and DNA replication following multiple administrations of dimethylnitrosamine (DMN) and to determine if these events were correlated with hepatotoxicity. Male CD-1 mice, 50-100 days old, were dosed daily, p.o., with DMN in water at dose levels of 2, 4, 7 and 10 mg/kg for 2 weeks. After 2, 7 and 14 days of dosing, hepatocytes were isolated by an in situ perfusion procedure, incubated in the presence of [3H] thymidine, and fixed. Unscheduled as well as scheduled DNA synthesis were assessed by quantitative autoradiography. Unscheduled DNA synthesis (UDS) represents DNA repair while scheduled DNA synthesis (S phase) represents DNA replication. In addition, the animals' serum was examined for enzymes which indicate hepatic toxicity. After 1, 7 and 14 days of dosing, animals were orbital-bled and the serum was analyzed for serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT), alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT). No morbidity or mortality was observed at dose levels of 2 and 4 mg/kg, but all animals receiving 7 and 10 mg/kg died after 4-6 days of dosing. GGT or AP were not elevated at any dose level or at any time point examined. At 4 mg/kg only a slight increase (less than or equal to 2 X) in the concentration of SGOT and SGPT was observed but a sharp increase (greater than 20 X) in replicative DNA synthesis was seen. The 2 mg/kg dose level of DMN did not increase replicative DNA synthesis and SGOT and SGPT were not elevated above control values at any time point following dosing at 2 mg/kg. A weakly positive DNA repair response was observed for dose levels of 4, 7 and 10 mg/kg DMN after two consecutive days of dosing. No DNA repair was observed after either 7 or 14 days of dosing at the 2 and 4 mg/kg/day levels. These results indicate that hepatic toxicity is associated with the induction of replicative DNA synthesis (S phase) but not with the induction of DNA repair. The results also confirm and extend a previous study (Doolittle et al., 1987b) indicating that a significant elevation in hepatic DNA replication is induced by hepatocarcinogens after multiple administrations of dose levels which do not alter hepatic DNA replication after a single administration. This finding indicates that the utility of the in vivo-in vitro hepatocyte assay may be enhanced by using a multi-dose protocol.

Animals↗
PubMedDate not supplied

[Effect of zinc-metallothionein on DNA replicative synthesis and single-stranded DNA-binding protein (SSB-protein) content in chromatin of mice bone marrow cells].

It was shown that the incubation in vitro of mice bone marrow cells with rat hepatic zinc-metallothionein (10-138 micrograms per 10(7) nuclear cells) leads to simultaneous 1.2-1.4 fold increase of DNA replicative synthesis level and 1.2-3 fold increase of the content of single-stranded DNA-binding proteins in chromatin of nuclear cells. Possible mechanisms of these effects connected with zinc ions donation and the increase of the content of DNA with single-stranded regions were discussed.

Animals↗
PubMed1999

Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA. Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate.

The T4 gp59 protein is the major accessory protein of the phage's replicative DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by promoting its assembly onto single-stranded (ss) DNA covered with cooperatively bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this helicase loading mechanism. Here, we characterize the properties of gp59-gp32-ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occupy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 species lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA and (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domain of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organizes gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes. Our results support a model in which gp59-gp32 interactions are non-essential for the co-occupancy of both proteins on ssDNA but are essential for the formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.

Bacteriophage M13↗
PubMed1997

Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.

Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences. Binding of Tus to a Ter site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion. In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication. In vitro competition assays demonstrated that base pair substitutions at positions 8-19 had significant effects on the free energy of Tus binding (DeltaDeltaG0 of 1.5 to >4. 0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo. Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Ter interactions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence.

Bacterial Proteins↗
PubMed1986

Inhibition of DNA replication by ozone in Chinese hamster V79 cells.

DNA replication in Chinese hamster lung fibroblasts, line V79, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. When the cells were exposed for 1 h at concentrations up to 6 ppm, the rate of DNA replication, as measured by [3H]thymidine incorporation, declined further during a 3-h period immediately following exposure. At higher ozone concentrations, at which more than 99.9% of the cells were killed, no further decline in DNA replication was seen beyond that immediately following exposure. Cultures exposed for 1 h to 10 mM ethyl methanesulfonate or to 10 J/m2 of ultraviolet (UV) light showed a similar progressive decline in the rate of DNA replication. The inhibition of DNA replication by ozone resembled that seen after exposure of cells to chemical mutagens or radiation and did not resemble the inhibition produced by metabolic poisons. The results may indicate that ozone or its reaction products interact directly with DNA in a way that inhibits replication.

Animals↗
PubMed1994

The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant ts4149 is, at the nonpermissive temperature, severely impaired in its ability to replicate its DNA genome. Compared to wild type, the amount of replication is suppressed by several orders of magnitude, and the little DNA that is replicated is not converted to mature linear genomes. We have demonstrated that this "DNA-" phenotype is not the result of a failure to produce early proteins. In agreement with the DNA- phenotype, intermediate and late gene expression were not detected. Marker rescue and DNA sequencing located the mutation in ts4149 to open reading frame D4. This gene has recently been shown to encode a 25-kDa protein with uracil DNA glycosylase activity (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden (1993), J. Virol. 67, 2503-2512). We speculate on the function of this "essential" viral repair enzyme and its role(s) in viral DNA replication.

Amino Acid Sequence↗
PubMed2005

Regeneration of the replication-associated proteins tandem direct repeat recognition nucleotide sequence at the origin of DNA replication of porcine circovirus type 1.

Four copies of a hexanucleotide (H) sequence are located to the right of the palindrome at the origin of DNA replication of the porcine circovirus type 1 (PCV1) genome. These sequences are organized in two direct tandems, the proximal H1/H2 and the distal H3/H4 repeats, and they have been shown to be binding sites for the essential Rep and Rep' proteins. Previous work demonstrated that infectious PCV1 virion can accommodate a variable number of H sequences at the origin of DNA replication. In this work, mutational analysis was conducted to elucidate the critical core element within the hexanucleotide with respect to self-DNA replication and progeny virus synthesis. It was found that while a single H sequence abutting the palindrome is sufficient for PCV1 viability, a tandem repeat arrangement is the more stable and thus preferred configuration. Within the H sequence, selected nucleotides at specific positions are critical for Rep-associated protein recognition and for viral DNA replication.

Animals↗
PubMed1997

Mammalian cell DNA replication.

The precise mechanisms involved in the regulation of the mammalian cell DNA-synthesizing machinery are poorly understood. In vitro DNA replication systems, in particular the employment of the simian virus 40 (SV40)-based cell-free DNA replication system, has identified several mammalian enzymes and proteins required for DNA synthesis. Although these proteins have been identified as playing a role in DNA replication, their functional organization allowing for the efficient replication of DNA has not been well defined. This review describes the proteins that have currently been defined as having a role in mammalian DNA replication and their proposed mechanisms of action. How these proteins may organize themselves to form multiprotein complexes, or larger DNA replication factories, allowing for efficient chromosomal DNA synthesis is discussed. In addition, the cell cycle regulation of mammalian DNA synthesis and the current status concerning mammalian DNA replication origins is described.

Animals↗
PubMed1998

DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon. It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. RESULTS: We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. CONCLUSION: These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.

Amino Acid Sequence↗
PubMed1994

Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

Adenosine Monophosphate↗
PubMed1981

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.

Bacteriophage phi X 174↗
PubMed2001

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system.

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.

DNA Helicases↗
PubMed2000

The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication.

Exposure of cells to ionizing radiation inhibits DNA replication in a dose-dependent manner. The dose response is biphasic and the initial steep component reflects inhibition of replicon initiation thought to be mediated by activation of the S-phase checkpoint. In mammalian cells, inhibition of replicon initiation requires the ataxia telagiectasia mutated ( ATM ) gene, a member of the phosphatidyl inositol kinase-like (PIKL) family of protein kinases. We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation. Exposure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibited replicon initiation in a time-dependent manner. Inhibition was at a maximum 1 h after irradiation and recovered at later times. Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for recovery, or only limited recovery, was observed for up to 8 h after irradiation. In addition a defect was observed in the maturation of nascent DNA. Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h. Normal kinetics of recovery were observed in xrs-5 cells, deficient in Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these cell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells may not be attributed entirely to the prolonged presence of unrepaired DNA dsb. We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in normal cells facilitates recovery of DNA replication after irradiation.

Animals↗
PubMed2004

Werner syndrome protein, the MRE11 complex and ATR: menage-à-trois in guarding genome stability during DNA replication?

The correct execution of the DNA replication process is crucially import for the maintenance of genome integrity of the cell. Several types of sources, both endogenous and exogenous, can give rise to DNA damage leading to the DNA replication fork arrest. The processes by which replication blockage is sensed by checkpoint sensors and how the pathway leading to resolution of stalled forks is activated are still not completely understood. However, recent emerging evidence suggests that one candidate for a sensor of replication stress is ATR and that, together with a member of RecQ family helicases, Werner syndrome protein (WRN) and MRE11 complex, can collaborate to promote the restarting of DNA synthesis through the resolution of stalled replication forks. Here, we discuss how WRN, the MRE11 complex and the ATR kinase could work together in response to replication blockage to avoid DNA replication fork collapse and genome instability.

DNA Damage↗