Peter A Dijkwel

Peter A Dijkwel
University of Virginia | UVa · Department of Biochemistry, Molecular Biology and Genetics

Ph.D.

About

49
Publications
1,307
Reads
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2,631
Citations
Additional affiliations
September 1985 - June 2011
University of Virginia
Position
  • Associate Professor of Research
Description
  • (Initiation of ) DNA Replication in Higher Eukaryotes, Amplification, Nuclear Structure
July 1981 - July 1985
Radboud Universiteit Nijmegen/KWF
Position
  • DNA Replication and the Nuclear Matrix in Cancer Cells
July 1980 - July 1981
Raad van Advies voor het Wetenschapsbeleid
Position
  • Mobility and Scientific Productivity
Education
September 1967 - June 1974
Utrecht University
Field of study
  • (Bio)chemistry

Publications

Publications (49)
Article
Full-text available
Studies in our laboratory over the last three decades have shown that the Chinese hamster dihydrofolate reductase (DHFR) origin of replication corresponds to a broad zone of inefficient initiation sites distributed throughout the spacer between the convergently transcribed DHFR and 2BE2121 genes. It is clear from mutational analysis that none of th...
Article
Full-text available
In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a st...
Article
The Chinese hamster dihydrofolate reductase (DHFR) origin consists of many inefficient initiation sites scattered throughout a 55-kb intergenic spacer, with the ori-beta, ori-beta', and ori-gamma subregions being preferred. To test for the presence of genetic replicators within these subregions, fragments containing ori-beta, ori-beta', or, as a ne...
Article
Full-text available
Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single co...
Article
Full-text available
The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-β, ori-β′, and ori-γ). We previously showed that deletion of the most active site or region (ori-β) has no demonstrable effect...
Article
Full-text available
Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred....
Article
Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate...
Article
Two independent two-dimensional agarose gel electrophoresis methods have been used to map the origin of replication that directs amplification of the C3 DNA puff of Rhynchosciara americana. The results of neutral/neutral two-dimensional gel electrophoresis show that DNA replication initiates at multiple sites in a zone of at least 6 kb situated imm...
Article
The neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques are sensitive physical mapping methods that have been used successfully to identify replication initiation sites in genomes of widely varying complexity. We present detailed methodology for the preparation of replication intermediates from mammalian cells and th...
Article
Full-text available
An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by ∼50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damagesensing (SDS) pathway appears to function primarily at the level of individual origins...
Article
In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is preferred over other start sites. To test the hypothesis that ori-beta contains a genetic replicator, we restored a deletion in the 3' end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream or...
Article
Full-text available
There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have b...
Article
Neutral/neutral two-dimensional gel electrophoresis is a sensitive physical mapping technique that has been successfully used to unambiguously identify replication initiation sites in genomes of widely varying complexity in vivo. The technique exploits the fact that restriction fragments containing different classes of replicative intermediates (si...
Article
Full-text available
Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much...
Article
Jacob and Brenner proposed a model for control of DNA replication in which a trans-acting initiator protein binds to a cis-acting replicator to effect initiation of nascent DNA chains at a fixed locus. Although replicators have been identified in prokaryotic and simple eukaryotic genomes, it has been much more difficult to demonstrate their presenc...
Article
In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete chromosomal locations. At each initiation site, a single small replication bubble is generated, which subsequently expands at Y-like replication forks. We wanted to know whether other eukaryotic organisms utilize similar initiation mechanisms. For this p...
Article
Full-text available
Previous two-dimensional gel replicon-mapping studies on the amplified dihydrofolate reductase (DHFR) domain in CHOC 400 cells suggested that replication can initiate at any of a large number of sites scattered throughout a 55-kb region lying between two convergently transcribed genes. It could be argued that this unusual distributive initiation mo...
Article
Establishing whether DNA replication in higher eukaryotic cells is regulated by genetic replicators has been one of the more challenging problems in cell biology. Several important replicon-mapping techniques have been developed in the past decade that have opened up new windows on replication origins. In the past few years, the application of thes...
Article
The eukaryotic genome appears to be organized in a loopwise fashion by periodic attachment to the nuclear matrix. The proposal that a chromatin loop corresponds to a functional domain has stirred interest in the properties of the DNA sequences at the bases of these loops, the matrix-attached regions (MARs). Evidence has been presented suggesting th...
Article
Full-text available
In previous studies, we utilized a neutral/neutral two-dimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lysing between the DHFR...
Article
Full-text available
Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and sy...
Article
Excerpt In the simple chromosomes of bacteria and their viruses, initiation of replication is controlled by a molecular switch consisting of a cis-regulatory genetic element (originally termed a “replicator”; Jacob and Brenner 1964) and a trans-acting, sequence-specific DNA-binding protein or protein complex (termed an “initiator”; Jacob and Brenne...
Article
Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a approximately 50 kb "initiation locus" (although the level of initiation detected at any given site within this region was relat...
Article
Full-text available
An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle travers...
Article
Full-text available
In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-...
Article
An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle travers...
Article
In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-...
Article
Full-text available
Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amp...
Article
Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amp...
Article
Several new methods have been used to localize replication initiation sites in mammalian chromosomes. The results of these studies argue strongly for the presence of defined sequence elements that function much like the origins in the genomes of simple microorganisms. However, relatively disparate results from in vivo and in vitro studies suggest t...
Article
Full-text available
This chapter discusses the aberrant DNA sequence amplification processes that occur in mammalian cells because of the clinical relevance to drug resistance and oncogene amplification in tumors and because the underlying mechanisms seem close to being understood at the molecular level. What seemed to be a relatively esoteric mutational phenomenon is...
Article
We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in se...
Article
Full-text available
It has been proposed that DNA in eukaryotic cells is synthesized via replication complexes that are fixed to a proteinaceous nuclear matrix. This model has not been universally accepted because the matrix and its associated DNA are usually prepared under hypertonic conditions that could facilitate non-specific aggregation of macromolecules. We ther...
Article
Full-text available
Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell li...
Article
The size of the looped, matrix-attached DNA domains was estimated in both nontransformed and transformed hamster cells. In BHK cells it was observed that in interphase as well as during mitosis, transformed cells, on the average, have shorter loops than nontransformed cells. In CHO cells, however, no alteration of the length of the looped DNA domai...
Article
In this paper we show that it is theoretically impossible to draw empirically founded conclusions about the relation between age and productivity. Only the relation between age and productivityincrease can be verified empirically. With this limitation in mind, a subsequent analysis of productivity data of Dutch physicists, chemists en economists, i...
Article
The main aim of this study is to estimate to what extent the productivity of researchers is influenced by their mobility. Based on emperical data of Dutch scientists it is shown that job mobility is a characteristic of productive scientists rather than a means to enhance productivity. Field mobility appears to stimulate productivity in the long run...
Article
Full-text available
Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from BHK nuclei by extraction with 2M-NaCl. The matrices were found to impose at least two levels of structural order upon nuclear DNA. From sedimentation studies it was inferred that metal depletion of the salt-extracted nuclei generated matrix structures, which s...
Article
Full-text available
The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted BHK-nuclei, prepared on covers lips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix....
Article
Full-text available
In a transformed cell line, derived from baby hamster kidney cells by treatment with ethylnitrosourea, degradation of DNA in isolated nuclei by endogenous nuclease was studied. Compared to the nontransformed cell line, the nuclear DNA of the transformed cells was found to be degraded to a much greater extent. This was reflected by a markedly lower...
Article
We have digested nuclei, isolated from [3H] thymidine pulse labelled cells, with nuclease S1. Short pulse labelled DNA fragments were excised by the enzyme and released upon subsequent treatment with 2 M NaCl. Only a small fraction of the label was released from the S1 digested nuclei by 0.5 M NaCl indicating that the cleavage sites were located in...
Article
The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine liver cells. At low daunomycin concentrations (1 and 2 muM) the rate of [3H]thymidine incorporation decreased progressively with the duration of exposure to the inhibitor. This was accompanied by a shift of nascent DNA intermediates of replicon size to...
Article
Full-text available
The attachment of replicating DNA to a rapidly sedimenting nuclear structure was investigated by digestion with various nucleases. When DNA was gradually removed by DNase I, pulse label incorporated during either 1 min or during 1 hour in the presence of arabinosylcytosine, remained preferentially attached to the nuclear structure. Single strand sp...
Article
Arabinosylcytosine at a 1 . 10(-4) molar concentration inhibited thymidine incorporation into DNA by more than 95%. In sucrose gradients the labelled dThd was predominantly found in short DNA chains. Labelled arabinosylcytosince (aC) was incorporated into DNA, as was labelled dThd, indicating that it causes a preferential inhibition of the chain po...

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