Methods in molecular biology (Clifton, N.J.), 2009
In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it ... more 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 standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsor...
Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofol... more 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 relatively low). This result contrasts markedly with data from an in vitro strand switching assay suggesting that > 80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiation per se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of 3H-thymidine moves through the structures observed on 2-D gels with the kinetics expected of bonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.
We have used two complementary two-dimensional gel electrophoretic methods to localize replicatio... more 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 several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.
The eukaryotic genome appears to be organized in a loopwise fashion by periodic attachment to the... more 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 that certain MARs act as boundary elements isolating domains from their chromosomal context. MARs have also been found in the vicinity of promoters and enhancers and they could act by displacing these cis-regulatory elements into the proper nuclear subcompartment. Attachment to the matrix might also play a role in DNA replication. A large body of evidence indicates that replication occurs on the nuclear matrix. This implies that any DNA sequence will be attached to the matrix at a certain time during the cell cycle. This transient mode of attachment contrasts with the proposed permanent attachment of origins of DNA replication with the nucle...
Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from BHK nuc... more 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 sedimented at significantly lower rates than control matrices. Fluorescence microscopy revealed that the reduced sedimentation rate is a consequence of the increase in the radius of the DNA halo, i.e. the DNA loops emanating from the residual nucleus. Addition of Cu ions to nuclei prior to salt extraction was found to induce contraction of this DNA halo. These results indicate that Cu ions may play an important role in stabilizing one level of DNA folding. When metal depletion had been brought about by thiol agents, a second effect was observed to occur. Within 15 min, salt-extracted nuclei disintegrated, generating irregularly shaped, slow...
The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine ... more 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 higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells. At high daunomycin concentrations (12 muM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [3H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre-fork mode of DNA synthesis.
Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV... more 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 synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, follo...
Publisher Summary This chapter discusses the aberrant DNA sequence amplification processes that o... more Publisher Summary 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 now a major determining factor in the genesis of cancer, as well as a serious deterrent to successful chemotherapeutic drug treatment regimens. The chapter traces the history of the discovery of DNA sequence amplification, cites several examples, and discusses the similarities and differences among these systems. Important methodologies developed for studying amplified sequences are also presented in the chapter. It reviews the viable models that attempt to explain the phenomenon in mammalian cells and shows the way the resolution of the underlying molecular mechanisms of amplification promises to teach much about both normal and aberrant chromosome dynamics (e. g., replication, recombination and repair processes, and chromosomal breakage and healing).
In a transformed cell line, derived from baby hamster kidney cells by treatment with ethylnitroso... more 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 proportion of DNA attached to the nuclear protein matrix in the transformed compared to the nontransformed cells. These observations can be accounted for by assuming that the chromatin of the transformed cell line has a conformation different from that of the nontransformed cells.
The position of replication origins and replication forks relative to the nuclear matrix was anal... more The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, 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. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.
Several new methods have been used to localize replication initiation sites in mammalian chromoso... more 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 that initiation reactions in mammalian chromosomes may have unique features, possibly related to a more complicated chromosomal architecture.
In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete c... more 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 purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.
Generally speaking, two kinds of mobility can be discerned: job mobility (a switch from one job t... more Generally speaking, two kinds of mobility can be discerned: job mobility (a switch from one job to another) and field mobility (a switch from one research field to another; such a switch can but does not necessarily have to be combined with a job switch). Indications of effects of job ...
In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is... more 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 ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.
Methods in molecular biology (Clifton, N.J.), 2009
In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it ... more 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 standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsor...
Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofol... more 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 relatively low). This result contrasts markedly with data from an in vitro strand switching assay suggesting that > 80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiation per se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of 3H-thymidine moves through the structures observed on 2-D gels with the kinetics expected of bonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.
We have used two complementary two-dimensional gel electrophoretic methods to localize replicatio... more 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 several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.
The eukaryotic genome appears to be organized in a loopwise fashion by periodic attachment to the... more 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 that certain MARs act as boundary elements isolating domains from their chromosomal context. MARs have also been found in the vicinity of promoters and enhancers and they could act by displacing these cis-regulatory elements into the proper nuclear subcompartment. Attachment to the matrix might also play a role in DNA replication. A large body of evidence indicates that replication occurs on the nuclear matrix. This implies that any DNA sequence will be attached to the matrix at a certain time during the cell cycle. This transient mode of attachment contrasts with the proposed permanent attachment of origins of DNA replication with the nucle...
Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from BHK nuc... more 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 sedimented at significantly lower rates than control matrices. Fluorescence microscopy revealed that the reduced sedimentation rate is a consequence of the increase in the radius of the DNA halo, i.e. the DNA loops emanating from the residual nucleus. Addition of Cu ions to nuclei prior to salt extraction was found to induce contraction of this DNA halo. These results indicate that Cu ions may play an important role in stabilizing one level of DNA folding. When metal depletion had been brought about by thiol agents, a second effect was observed to occur. Within 15 min, salt-extracted nuclei disintegrated, generating irregularly shaped, slow...
The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine ... more 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 higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells. At high daunomycin concentrations (12 muM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [3H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre-fork mode of DNA synthesis.
Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV... more 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 synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, follo...
Publisher Summary This chapter discusses the aberrant DNA sequence amplification processes that o... more Publisher Summary 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 now a major determining factor in the genesis of cancer, as well as a serious deterrent to successful chemotherapeutic drug treatment regimens. The chapter traces the history of the discovery of DNA sequence amplification, cites several examples, and discusses the similarities and differences among these systems. Important methodologies developed for studying amplified sequences are also presented in the chapter. It reviews the viable models that attempt to explain the phenomenon in mammalian cells and shows the way the resolution of the underlying molecular mechanisms of amplification promises to teach much about both normal and aberrant chromosome dynamics (e. g., replication, recombination and repair processes, and chromosomal breakage and healing).
In a transformed cell line, derived from baby hamster kidney cells by treatment with ethylnitroso... more 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 proportion of DNA attached to the nuclear protein matrix in the transformed compared to the nontransformed cells. These observations can be accounted for by assuming that the chromatin of the transformed cell line has a conformation different from that of the nontransformed cells.
The position of replication origins and replication forks relative to the nuclear matrix was anal... more The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, 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. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.
Several new methods have been used to localize replication initiation sites in mammalian chromoso... more 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 that initiation reactions in mammalian chromosomes may have unique features, possibly related to a more complicated chromosomal architecture.
In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete c... more 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 purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.
Generally speaking, two kinds of mobility can be discerned: job mobility (a switch from one job t... more Generally speaking, two kinds of mobility can be discerned: job mobility (a switch from one job to another) and field mobility (a switch from one research field to another; such a switch can but does not necessarily have to be combined with a job switch). Indications of effects of job ...
In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is... more 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 ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.
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