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M L DePamphilis

Publications and source records attributed to M L DePamphilis.

At least 55 records · Page 3Linked to original sources
PubMed1993

Origins of DNA replication that function in eukaryotic cells.

This past year has seen a significant increase in our understanding of eukaryotic origins of replication, of the proteins that identify these origins, of DNA sequences that promote their unwinding, and of transcription factors that stimulate origin activity. DNA replication begins at specific sites in both simple and complex genomes, but origins in complex genomes may include nuclear structure as well as DNA sequence.

Animals↗
PubMed1993

Site-specific initiation of DNA replication in metazoan chromosomes and the role of nuclear organization.

We have asked whether or not Xenopus eggs or egg extracts, which have previously been shown to replicate essentially any DNA molecule, will preferentially utilize a known mammalian OBR. Our results reveal that Xenopus egg extracts can preferentially initiate DNA replication at sites chosen in vivo by the hamster cell, provided that the DNA substrate is presented to the extract in the form of a nucleus rather than bare DNA. Thus, site-specific initiation of DNA replication in metazoan cell chromosomes appears to be determined by nuclear organization as well as DNA sequence. We have also considered whether or not BPV, which was previously reported to regulate its copy number through negative as well as positive cis-acting sequences, provides a suitable paradigm for cellular origins. The BPV genome was found to contain cis-acting sequences that can suppress DNA replication driven by a lytic virus such as PyV. However, this suppression did not require any BPV protein, did not limit PyV origin activity to one initiation event per S phase, and did not affect BPV origin activity. These results, together with data from other laboratories, strongly suggest that BPV is simply a slow-replicating version of SV40 and PyV and therefore is not an appropriate model to explain how initiation of cellular DNA replication is limited to once per cell cycle.

Animals↗
PubMed1992

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

Antigens, Polyomavirus Transforming↗
PubMed1992

Guide to identification of origins of DNA replication in eukaryotic cell chromosomes.

Several experimental approaches for identification of origins of DNA replication have been developed recently that allow, for the first time, unique initiation sites in mammalian chromosomes to be mapped at single-copy loci. A brief description of the rationale, advantages, and limitations has been provided for each approach, as well as information that can help the reader choose the method(s) most suitable for a particular system. The various methods are divided into three groups: (1) analysis of nascent DNA strands, (2) analysis of DNA structures, and (3) analysis of origin activity (i.e., ability to support autonomous replication). It is hoped that this information will serve as a practical guide for identifying new origins of replication.

Animals↗
PubMed1991

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

Animals↗
PubMed1991

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities.

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.

Amanitins↗
PubMed1991

T-antigen binding to site I facilitates initiation of SV40 DNA replication but does not affect bidirectionality.

SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.

Antigens, Polyomavirus Transforming↗
PubMed1990

Identification of an origin of bidirectional DNA replication in mammalian chromosomes.

Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.

Animals↗
PubMed1990

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.

Animals↗
PubMed1990

Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.

The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.

Animals↗
PubMed1990

Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

Animals↗
PubMed1989

Sequences that promote formation of catenated intertwines during termination of DNA replication.

The normal sequence at which SV40 DNA replication terminates (TER) is unusual in that it promotes formation of catenated intertwines when two converging replication forks enter to complete replication (Weaver et al., 1985). Here we show that yeast centromeric sequences also exhibit this phenomenon. CEN3 caused accumulation of late replicating intermediates and catenated dimers in plasmids replicating in mammalian cells, but only when it was located in the termination region (180 degrees from ori), and only when cells were subjected to hypertonic shock to reduce topoisomerase II activity. Therefore, formation of catenated intertwines during termination of DNA replication was sequence dependent, suggesting that topoisomerase II acts behind replication forks in the termination region to remove intertwines generated by unwinding DNA rather than acting after replication is completed and catenates are formed. Under normal physiological conditions, CEN3 did not promote formation of catenated dimers in either mammalian or yeast cells. Therefore, CEN does not maintain association of sister chromatids during mitosis in yeast by introducing stable catenated intertwines during replication.

Cell Cycle↗
PubMed1989

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation F101, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1:67:21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.

Animals↗
PubMed1989

Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos.

Undifferentiated mouse embryonal carcinoma (EC) cells are capable of transactivating the adenovirus EIIa promoter in the absence of its normal transactivator, E1A protein, suggesting that EC cells contain an E1A-like activity. In an effort to identify where this activity appears during normal mouse development, mouse oocytes and preimplantation embryos were injected with plasmids containing the EIIa promoter coupled to various reporter genes. These expression vectors were fully active in human 293 cells where E1A is present, but were inactive in differentiated fibroblast cell lines unless cotransfected with the E1A gene. In mouse oocytes and preimplantation embryos, EIIa promoter activity in the absence of adenovirus E1A protein was equivalent to or greater than activity of the HSV thymidine kinase promoter coupled to a strong enhancer. Coinjection of the E1A gene failed to stimulate EIIa activity further, perhaps because c-myc protein, which has been reported to transactivate this promoter, was already present at high levels in mouse oocytes. Activation of the EIIa promoter in the absence of E1A was unique to mouse oocytes and preimplantation embryos because gene expression from an EIIa promoter introduced into transgenic mice was observed only in the adult ovary, and particularly in the oocytes. In addition, post-implantation transgenic embryos failed to express the E1A-activatable reporter gene, thereby indicating that expression from the EIIa promoter is restricted to the relatively undifferentiated stages of oogenesis and preimplantation development. These data suggest that cellular promoters of the class that can be transactivated by E1A may serve uniquely to initiate transcription of genes that are needed for preimplantation development.

Adenovirus Early Proteins↗