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J A Huberman

Publications and source records attributed to J A Huberman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Large, complex modular structure of a fission yeast DNA replication origin.

BACKGROUND: In the budding yeast, Saccharomyces cerevisiae, each DNA replication origin is associated with an autonomously replicating sequence (ARS) element. Each element contains several modules, including an essential close match to the 11 base-pair (bp) ARS consensus sequence (ACS) and two or three short (< 20 bp) stimulatory motifs, within a stretch of approximately 150 bp or less. To determine whether a similar origin structure exists in the evolutionarily distant fission yeast, Schizosaccharomyces pombe, we used deletion and linker substitution scanning to identify the sequences important for the function of ars3002, a chromosomal replication origin. RESULTS: We detected two large (30-55 bp) essential regions and several additional stimulatory sequences within a 600 bp stretch of a restriction fragment containing ars3002. The two essential regions are similar to each other, and sequences similar to them are found in all known S. pombe ARS elements, suggesting that one or both of them may represent the S. pombe equivalent of the S. cerevisiae ACS. CONCLUSIONS: Like S. cerevisiae origins, the S. pombe origin, ars3002, possesses a modular structure, but the number and size of modules is greater for ars3002, and ars3002 is larger than S. cerevisiae origins. These observations suggest that origin function in S. pombe requires more protein-DNA interactions than in S. cerevisiae.

Base Sequence↗
PubMed1996

Topological complexity of SV40 minichromosomes.

During attempts to measure the extent to which the proteins of simian virus 40 (SV40) minichromosomes restrain the ability of SV40 DNA to alter its twist in response to temperature changes, we found that temperature-shift-induced linking number changes are not reversible for isolated minichromosomes, suggesting that such changes, both in isolated minichromosomes and in cells, may be a consequence of structural alterations in chromatin proteins rather than of simple changes in DNA twist. We also found that the SV40 minichromosome pool is composed of subpopulations that display different responses to temperature shifts. For example, the linking number of DNA in newly replicated minichromosomes is more responsive to in vivo temperature changes than is the linking number of DNA in bulk minichromosomes. In addition, the linking number profiles of both isolated and intracellular minichromosomes change during the course of infection. These observations emphasize the topological complexity of SV40 minichromosomes and encourage caution in the interpretation of experiments carried out on bulk minichromosomes.

DNA Replication↗
PubMed1996

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

Amino Acid Sequence↗
PubMed1996

Cell cycle control of S phase: a comparison of two yeasts.

The mechanisms responsible for correct timing of DNA synthesis within the cell cycle and for limiting replication to one round per cell cycle are basically similar in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite many differences in detail. In both cases, the timing of initiation and the prevention of additional rounds are controlled by the activity levels of B-type cyclins. These similarities are likely to extend to other eukaryotic organisms.

Cell Cycle↗
PubMed1995

pDblet, a stable autonomously replicating shuttle vector for Schizosaccharomyces pombe.

We have constructed a new multipurpose stable shuttle vector for the fission yeast Schizosaccharomyces pombe (Sp). Plasmid pDblet was designed to provide convenient features for molecular work and to overcome the inconveniences of previously designed Sp vectors. It contains the Sp ura4 gene as selectable marker and a new highly efficient ARS (autonomously replicating sequence) element, allowing the vector to remain stable as a monomer in Sp. In addition, pDblet transforms Sp with high efficiency and has high mitotic stability and low copy number.

DNA Replication↗
PubMed1995

Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels.

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.

Animals↗
PubMed1995

Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit.

Animals↗
PubMed1994

Three ARS elements contribute to the ura4 replication origin region in the fission yeast, Schizosaccharomyces pombe.

The ura4 replication origin region, which is located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains multiple initiation sites. We have used 2D gel electrophoretic replicon mapping methods to study the distribution of these initiation sites, and have found that they are concentrated near three ARS elements (stretches of DNA which permit autonomous plasmid replication). To determine the roles of these ARS elements in the function of the ura4 origin region, we deleted either one or two of them from the chromosome and then assessed the consequences of the deletions by 2D gel electrophoresis. The results suggest that each of the three ARS elements is responsible for the initiation events in its vicinity and that the ARS elements interfere with each other in a hierarchical fashion. It is possible that the large initiation zones of animal cells are similarly composed of multiple mutually interfering origins.

Chromosome Mapping↗
PubMed1994

Comparison of the two major ARS elements of the ura4 replication origin region with other ARS elements in the fission yeast, Schizosaccharomyces pombe.

We have previously reported that the replication origin region located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains three closely spaced origins, each associated with an autonomously replicating sequence (ARS) element. Here we report the nucleotide sequences of two of these ARS elements, ars3002 and ars3003. The two ARS elements are located on either side of a transcribed 1.5 kb open reading frame. Like 11 other S. pombe ARS elements whose sequences have previously been determined in other laboratories, the 2 new ARS elements are unusually A+T-rich. All 13 ARS elements contain easily unwound stretches of DNA. Each of the ARS elements contains numerous copies, at a higher than expected frequency, of short stretches of A+T-rich DNA in which most of the Ts are on one strand and most of the As are on the complementary strand. We discuss the potential significance for ARS function of these multiple asymmetric A+T-rich sequences.

Base Composition↗
PubMed1992

Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe.

In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.

Chromosomes, Fungal↗
PubMed1992

Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.

Chromosomes, Fungal↗
PubMed1991

Cell cycle control of initiation of eukaryotic DNA replication.

Eukaryotic DNA replication is confined to a specific portion of the cell cycle (the S phase) and is highly regulated: every segment of the genome is replicated once per S phase, but no segment is normally replicated more than once. How this tight control of replication is accomplished is not known. However, the pace of research into the mechanisms of eukaryotic DNA replication and of cell cycle control has accelerated dramatically within the past few years. Recent investigations provide, for the first time, hints of how control of replication may be coupled at the molecular level to control of the cell cycle. This review is intended to bring these recent investigations to the reader's attention and to speculate about their relationships to each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗