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At least 91 records · Page 5Linked to original sources
PubMed1981

DNA polymerase accuracy and spontaneous mutation rates: frequencies of purine.purine, purine.pyrimidine, and pyrimidine.pyrimidine mismatches during DNA replication.

DNA from the am16 mutant of bacteriophage phi X174 may be replicated in vitro and expressed in vivo to give five classes of revertants. Each class may be specifically induced by the appropriate biasing of the concentrations of deoxynucleoside triphosphates in a predictable manner. The frequency of each reversion follows a kinetic rate equation relating it to the concentrations of the triphosphates involved in the substitution. The reversions corresponding to TAG leads to GAG, AAG, CAG, TGG, and TCG are calculated to occur with frequencies of 5 X 10(-7), 4 X 10(-7), 4 X 10(-7), approximately 2 X 10(-7), and approximately 5 X 10(-9), respectively, at the concentration of triphosphates found in vivo. The frequencies are in the range found for the reversion of the phage in vivo and so are consistent with errors in nucleotide selection by DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) III being largely responsible for the rate of spontaneous mutation in vivo. The relative frequency of mispairing leading to misincorporation is: purine.purine approximately purine.pyrimidine much greater than pyrimidine.pyrimidine, confirming predictions from model-building studies that transversions arise through purine.purine mismatches.

Bacteriophage phi X 174↗
PubMed1981

Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.

Cell Cycle↗
PubMed1966

Enzymatic mechanisms of DNA replication.

DNA polymerases purified from several sources are characterized by replication of the 3'-hydroxy-terminated strand of a helical template. Failure to achieve simultaneous replication of the 5'-strand leads to aberrations in the synthesized DNA, described as nondenaturability and branching. Aberrations in synthesized DNA were not observed when (a) the 5'-strand was destroyed by a specific nuclease during the course of replication or (b) a single-stranded (circular) phage (M13) DNA served as template. Replication of a single-stranded, circular DNA produced a helical product, but the nature of initiation of a new strand by the circular template remains to be explained. Hypothetical mechanisms for simultaneous replication of the 5'-strand are presented as is the possibility that the tertiary structure of the DNA, as for example, a circular form of the helix, is of prime importance in in vivo replication.

Bacteriophages↗
PubMed1975

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.

Adenoviridae↗
PubMed1978

DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons.

The direction of replication of DNA within replicons of Physarum polycephalum was studied by pulse-labelling with 5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) -photolysis and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within replicons at all stages of completion is split in two equal halves upon UV irradiation when BrdUrd was given at the time of initiation of DNA synthesis. This shows that replication within replicons of Physarum polycephalum starts at an origin located in the center of each unit, proceeding bidirectionally from this origin.

Bromodeoxyuridine↗
PubMed1997

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.

Amino Acid Sequence↗
PubMed1999

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.

Cell Cycle Proteins↗
PubMed1985

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.

Adenoviruses, Human↗
PubMed2006

Replication-dependent DNA damage response triggered by roscovitine induces an uncoupling of DNA replication proteins.

The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G(1) and in G(2) phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase delta, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.

Cell Proliferation↗
PubMed1983

DNA repair replication, DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro.

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10(-3) moles/l. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10(-6) moles/l). Fibroblasts exposed to AM at 10(-4) moles/l in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes. A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (less than 10(-7) moles/l). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G0 or G1. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles. These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.

Cell Cycle↗
PubMed1987

On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.

Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.

DNA Replication↗
PubMed1979

A ts mutant isolated from CHL cells: inhibition of DNA replication at nonpermissive temperature.

A mutant Chinese hamster cell clone has been isolated from an S phase culture. This clone, ts154, could not replicate DNA at nonpermissive temperature (39 degrees C), although it synthesized DNA normally at permissive temperature (33 degrees C). After transfer to 39 degrees, ts154 cells replicated DNA semiconservatively through precisely two complete rounds and divided twice before they ceased DNA synthesis and further growth. The mutation in ts154 affects DNA replication specifically, as its rates of cellular transcription, its ability to support growth of vesicular stomatitis virus, and its ribo- and deoxyribonucleoside triphosphate pools were normal at 39 degrees. Cultures of ts154 resumed DNA replication within 6h when shifted from 39 degrees to 33 degrees, even after 78 h at the nonpermissive temperature. Upon return to 33 degrees, mutant cells achieved a normal steady-state rate of DNA synthesis by 10 h, suggesting that they had come to rest within G1 phase at 39 degrees. Resumption of DNA synthesis and growth after a shift-down to 33 degrees were sensitive to cycloheximide in ts154, and thus likely required synthesis of new protein.

Animals↗
PubMed1995

The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.

Chromatin assembly factor I (CAF-I) from human cell nuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histones H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA.

Amino Acid Sequence↗
PubMed2001

Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae.

The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G(1)/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.

Cell Cycle Proteins↗