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

Hou, Zhenglin; Umthun, Angela R.; Dobbs, Drena

doi:10.1021/bi00014a011

Cited by 10 publications

(5 citation statements)

References 78 publications

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“…Such activity could bear on the enhancement effect reported here. Numerous single-stranded DNA or RNA binding proteins have been implicated in replication and transcription (25)(26)(27)(28)(29). However, NonO's enhancing activity is still apparent in the presence of single-stranded DNA competitors (data not shown).…”

Section: Discussionmentioning

confidence: 98%

NonO enhances the association of many DNA-binding proteins to their targets

Yang

Tucker

et al. 1997

Nucleic Acids Research

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NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

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Section: Discussionmentioning

confidence: 98%

NonO enhances the association of many DNA-binding proteins to their targets

Yang

Tucker

et al. 1997

Nucleic Acids Research

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“…Recently, a strand-specific type I element binding protein, ssA-TIBF, was identified by an in vitro gel shift assay (22,43). It is plausible that this single-stranded binding site is generated only during DNA replication.…”

Section: Discussionmentioning

confidence: 99%

Type I Elements Mediate Replication Fork Pausing at Conserved Upstream Sites in the Tetrahymena thermophila Ribosomal DNA Minichromosome

MacAlpine

Zhi

Kapler

1997

Molecular and Cellular Biology

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Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome. During vegetative growth, the rDNA is replicated exclusively from origins in the 5 nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5 NTS is not. Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5 NTS. Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required. A conserved tripartite sequence was identified at each pausing site. Mutations in type I elements diminish pausing at proximal RFPs. Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks. Studies with rDNA transformants revealed a strong directional bias for fork pausing. Strong pausing only occurred in forks moving toward the rRNA-coding region. We propose that fork pausing in the 5 NTS evolved to synchronize replication and transcription of the downstream rRNA genes.

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“…TIF1 Purification and Protein Sequencing-S100 extracts were prepared from strain CU428 as described (29). Approximately 60 ml of extract (protein concentration 8 -10 mg/ml) were obtained from a 4-liter culture of cells grown to a density of 2 ϫ 10 5 /ml.…”

Section: Methodsmentioning

confidence: 99%

“…The most extensively studied type I element binding activity, TIF1 (previously designated ssA-TIBF), is a homotetramer with a subunit molecular mass of 21 kDa (25,28,29). Purified TIF1 binds in a sequence-specific manner to single-stranded DNA corresponding to either the A-rich or T-rich strand of the type I elements, raising the possibility that it stabilizes replication origins in an unwound state (25).…”

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confidence: 99%

Cloning and Biochemical Analysis of the TetrahymenaOrigin Binding Protein TIF1

Saha

Nicholson

Kapler

2001

Journal of Biological Chemistry

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Cis-acting type I elements regulate the initiation of DNA replication, replication fork movement, and transcription of the Tetrahymena thermophila rDNA minichromosome and are required for cell cycle-controlled replication and developmentally programmed gene amplification. Previous studies identified three in vitro single-stranded type I element binding activities that were proposed to play distinct roles in replication control. Here we describe the cloning of one of these genes, TIF1, and we provide evidence for its association with type I elements in vivo. Furthermore, we show that TIF1 interacts (in vitro and in vivo) with pause site elements (PSE), which co-localize with replication initiation and fork arrest sites, and are shown to be essential. The in vivo accessibility of PSE and type I elements to potassium permanganate suggests that origin regions are frequently unwound in native chromatin. TIF1 contains sequence similarity to the Solanum tuberosum single strand-specific transcription factor, p24, and a related Arabidopsis protein. Antisense inhibition studies suggest that TIF1 competes with other proteins for PSE and type I element binding. TIF1 displays a marked strand bias in vivo, discriminating between origin-and promoter-proximal type I elements. We propose that this bias selectively modulates the binding of a different subset of proteins to the respective regulatory elements.

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

Abstract: Hou, Zhen; Umthun, Angela R.; and Dobbs, Drena L., "A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA" (1995). Zoology and Genetics Publications. 47.

Cited by 10 publications

References 78 publications

NonO enhances the association of many DNA-binding proteins to their targets

NonO enhances the association of many DNA-binding proteins to their targets

Type I Elements Mediate Replication Fork Pausing at Conserved Upstream Sites in the Tetrahymena thermophila Ribosomal DNA Minichromosome

Cloning and Biochemical Analysis of the TetrahymenaOrigin Binding Protein TIF1

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