Biomedical subjects

J L Hamlin

Publications and source records attributed to J L Hamlin.

At least 19 recordsLinked to original sources

PubMed2005

Promiscuous initiation on mammalian chromosomal DNA templates and its possible suppression by transcription.

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 negative control, a fragment from the DHFR gene that never initiates in loco were placed at ectopic chromosomal positions. Two-dimensional gel analysis demonstrates that initiation occurs in all three fragments, including the fragment from the gene, and appears to occur at different positions within each fragment in different cells in the population. However, initiation is not detectable in the adjacent, transcribed, neomycin-resistance marker. When a cosmid containing an active DHFR gene was inserted into an ectopic chromosomal site, initiation could no longer be observed in the body of the gene, but was clearly evident in the transcriptionally-silent bacterial vector sequences. In the thirteen independent chromosomal positions into which the four different constructs were inserted, initiation occurred in early S-phase. Since these loci necessarily had to be permissive for transcription of the neo(r) marker or DHFR gene used to select transfectants, it is possible that early-firing origin activity and local transcription are somehow linked. In total, these data suggest that potential initiation sites are distributed at very frequent intervals in the mammalian genome, with usage being regulated both positively and negatively by local transcription.

Animals↗

PubMed2003

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation.

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 copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Animals↗

PubMed2003

The dihydrofolate reductase origin of replication does not contain any nonredundant genetic elements required for origin activity.

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-beta, ori-beta', and ori-gamma). We previously showed that deletion of the most active site or region (ori-beta) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-beta', both ori-beta and ori-beta', an 11-kb region just downstream from the DHFR gene, or the central approximately 40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur.

Animals↗

PubMed2000

Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

Chromosome Aberrations↗

PubMed2000

Dispersive initiation of replication in the Chinese hamster rhodopsin locus.

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 reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells.

Animals↗

PubMed1999

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Animals↗

PubMed1999

Physical and genetic mapping of mammalian replication origins.

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 their analysis by both neutral/neutral and neutral/alkaline two-dimensional gel approaches. The methods described allow characterization of the replication pattern of single-copy loci, even in mammalian cells. When applied to metazoans, initiation is found to occur at multiple sites scattered throughout zones that can be as long as 50 kb, with some subregions being preferred. Although these observations do not rule out the possibility of genetically defined replicators, they offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in complex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb from the local initiation zone can markedly affect origin activity in the zone.

Animals↗

PubMed1999

DNA replication during amplification of the C3 puff of Rhynchosciara americana initiates at multiple sites in a 6 kb region.

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 immediately upstream from the promoter of the main transcription unit of this puff. The complementary neutral/alkaline two-dimensional gel electrophoresis technique shows that, within the initiation zone, forks move in both directions. In contrast, unidirectional fork movement away from the initiation zone is observed at the ends of the region, implying that it is the only place in the amplified region of the C3 puff where initiations occur. Since the initiation zone coincides with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism.

Animals↗

PubMed1998

Attachment to the nuclear matrix mediates specific alterations in chromatin structure.

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Animals↗

PubMed1998

Distal sequences, but not ori-beta/OBR-1, are essential for initiation of DNA replication in the Chinese hamster DHFR origin.

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.

Animals↗

PubMed1998

Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin.

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 been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-beta, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-beta and a second locus (ori-gamma) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-beta; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-beta and ori-gamma being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.

Animals↗

PubMed1997

The dual effect of mimosine on DNA replication.

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells, but the mechanism by which this inhibition is achieved is unknown. The drug has been proposed either to inhibit initiation at origins of replication or to inhibit chain elongation by lowering nucleotide pool levels. In an attempt to determine which mode of action is correct, we have analyzed its effects on SV40 DNA replication. Using a two-dimensional gel replicon mapping technique, we show that mimosine completely inhibits incorporation of [3H]thymidine into viral DNA, but only after approximately 4 h. Qualitative analysis of replication intermediates during this interval suggests that the drug partially inhibits both initiation and elongation, and pulse-chase experiments support this contention. The drug has no effect when added directly to an SV40 in vitro replication extract. However, extracts prepared from cells pretreated with mimosine are compromised in their ability to support replication in vitro in the presence of a full complement of nucleotides. Thus, although mimosine may alter nucleotide pool levels in vivo, it also appears to affect one or more essential replication proteins.

Animals↗

PubMed1997

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G1/S transition in Chinese hamster ovary cells.

In response to a moderate dose of radiation, asynchronous mammalian cell populations rapidly and transiently down-regulate the rate of DNA synthesis to approximately 50% of preirradiation values. We show here that only half of the reduction in overall replication rate can be accounted for by direct inhibition of initiation at origins in S-phase cells. The other half results from the operation of a newly defined cell cycle checkpoint that functions at the G1/S transition. This checkpoint senses damage incurred at any time during the last 2 hr of G1 and effectively prevents entry into the S period. The G1/S and S-phase checkpoints are both p53-independent and, unlike the p53-mediated G1 checkpoint, respond rapidly to radiation, suggesting that they may represent major damage-sensing mechanisms connecting the replication machinery with DNA repair pathways.

Animals↗

PubMed1997

Cloning and characterization of Chinese hamster p53 cDNA.

We have cloned and sequenced Chinese hamster p53 cDNA and have compared the p53 sequence in different Chinese hamster cell lines to several relevant phenotypes. Our results indicate that a mutation in CHO cells that changes Thr211 to Lys211 abrogates the ability to arrest in G1 and apparently renders cells capable of amplifying DNA. However, this mutation has no effect on the G2 checkpoint or on acute down-regulation of DNA replication after a radiation challenge.

Amino Acid Sequence↗

PubMed1997

Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis.

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 (single forks, initiation bubbles, or termination structures) migrate to different and characteristic positions in agarose gels. The replication pattern of any region of interest can then be determined by sequential hybridization with appropriate radioactive probes from that region.

Animals↗

PubMed1997

S phase damage sensing checkpoints in mammalian cells.

Mammalian cells have evolved multiple responses for dealing with DNA damage. One response is to acutely downregulate DNA synthesis at the initiation step. Essentially nothing is known about the initial signal that activates this SDS pathway or the macromolecules involved in transducing the signal into the final inhibitory step at origins. Determining whether any radiation induced changes in known proteins involved in cell cycle regulation or in other signal transduction pathways are primary or secondary responses to DNA damage constitutes a major challenge to identifying members of the pathway. It may turn out to be easier to identify the final mediator in the pathway, namely the protein(s) whose interaction with origins is ultimately affected by radiation. Hopefully, mutations in SDS genes in genetically tractable systems such as S cerevisiae or Schizosaccharomyces pombe will allow the identification of homologous genes in mammals. Most tumour cells are TP53 negative, and yet it is not clear that TP53 status influences radiation sensitivity. The SDS pathway may therefore represent an important protective mechanism that stands in the way of effective tumour cell killing by radiation therapy. It is hoped that an understanding of this pathway will provide opportunities for developing novel antineoplastic targets and/or radiation sensitizers.

Animals↗

PubMed1996

Mimosine targets serine hydroxymethyltransferase.

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of approximately 2.5 h. We have shown previously that [3H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly- cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly- cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.

Amino Acid Sequence↗

PubMed1996

Sequence and context effects on origin function in mammalian cells.

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 presence in mammalian chromosomes. Owing to the lack of genetic approaches for identifying mammalian replicators, investigators have directed attention to localizing nascent strand start sites, which should lie close to replicators. Toward this end, a variety of clever techniques have been invented for analyzing replication intermediates, but only rarely have more than one of these techniques been applied to a single locus. However, virtually all have been used to analyze the dihydrofolate reductase locus in CHO cells. The picture that has developed in this locus is that initiation can occur at any of a large number of sites scattered throughout a broad zone, but somewhat more frequently near two sites that may correspond to true genetic replicators. Furthermore, it appears that local transcriptional activity, as well as appropriate torsional stress (as imparted by local attachment to the nuclear matrix), may have profound effects on origin activity.

Animals↗