Biomedical subjects

D L Dobbs

Publications and source records attributed to D L Dobbs.

8 recordsLinked to original sources

PubMed1995

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

Animals↗

PubMed1995

A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA.

Type I repeat sequences are evolutionarily conserved sequence elements found in the replication origin and transcriptional promoter region of the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single-stranded DNA binding protein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence. Quantitative binding competition experiments performed with purified ssA-TIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent Kd = 3.0 x 10(-10) M). A mutation in the ssA-TIBF binding site previously shown to be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro. ssA-TIBF also binds with high affinity to a copy of the Type I repeat sequence within the essential promoter region defined by in vitro transcription assays. The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region. The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.

Animals↗

PubMed1995

Visualization of nucleosomal substructure in native chromatin by atomic force microscopy.

Intact rDNA minichromosomes from Tetrahymena thermophila were isolated as native chromatin and imaged by atomic force microscopy (AFM). AFM measurements of condensed rDNA chromatin were consistent with a 30 nm fiber that frequently (87% of molecules observed) contained stretches of nucleosome cores arranged in a zig-zag conformation. Examination of rDNA chromatin in a dispersed conformation by tapping mode AFM in low humidity resulted in high resolution images of partially dissociated nucleosome cores and associated linker DNA. A majority of these nucleosome cores contained six to eight smaller particles with dimensions consistent with those of individual histones. Many of the nucleosome cores showed a striking resemblance to the wedge (35%), axial (15%), and front (6%) views of the nucleosome histone octamer modeled by Arents et al. [Arents, G., Burlingame, R. W., Wang, B.-C., Love, W.E., & Moudrianakis, E. N. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10148-10152]. This direct visualization of histone subunits and nucleosomal substructure in native chromatin illustrates the potential use of AFM to localize individual proteins in condensed cellular chromatin.

Animals↗

PubMed1995

Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells of Xenopus laevis.

The plant amino acid mimosine has been reported to block cell cycle progression and DNA replication in cultured mammalian cells, perhaps by blocking initiation. In this study, we show that mimosine does not block initiation or any other step in DNA replication in embryonic cells of Xenopus laevis. Mimosine does not block DNA replication in cell-free "cycling" extracts of Xenopus eggs, nor does it block M to S phase transition in cell-free egg extracts released from metaphase arrest. Microinjection of mimosine into 4-cell embryos had no visible effect on development during the first 3 days after fertilization. Prior to the midblastula transition, when the cell cycle consists of alternating S and M phases, neither chromosomal DNA replication nor replication of microinjected plasmid DNA were inhibited by mimosine microinjected into cleaving Xenopus embryos. Microinjection of mimosine after the midblastula transition, when large endogenous stockpiles of DNA replication components have begun to be depleted and Xenopus embryonic cells have acquired G1 and G2 phases, still did not inhibit cell cycle progression or DNA replication. In marked contrast, mimosine arrested the growth of proliferating cultured Xenopus kidney epithelial A6 cells near the G1/S boundary. We conclude that mimosine appears to block DNA replication and cell cycle progression in somatic cells, but has no apparent effect in rapidly dividing Xenopus embryonic cells.

Animals↗

PubMed1995

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.

Animals↗

PubMed1994

Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.

An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.

Animals↗

PubMed1994

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.

Amino Acid Sequence↗

PubMed1994

Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes.

A novel DNA helicase isolated from Xenopus laevis ovaries [Poll, E. H. A., & Benbow, R. M. (1988) Biochemistry 27, 8701-8706] was characterized biochemically. The directionality of DNA unwinding was determined to be 3' to 5'. A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases. Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not. Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate. X. laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA. DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4. The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity. CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition. The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP.

Adenosine Triphosphatases↗