Single-stranded DNA Binding Factor Research Articles

The NssBF element, a sequence of the Drosophila melanogaster retrotransposon 1731 potentially implicated in transcriptional repression and replication

The nuclear single-stranded DNA binding factor (NssBF) has been characterized as a nuclear protein that binds to a 26 nucleotides sequence in the long terminal repeat (LTR) of the Drosophila melanogaster 1731 retrotransposon. This sequence, called NssBF element, was analysed by gel retardation experiments using wild-type and mutated oligonucleotides. In vitro transcription experiments were performed and suggest that NssBF element binding protein(s) represses transcription through the 1731 promoter. Furthermore, computer assisted sequence comparisons put forward a possible role of this element and/or its associated DNA binding proteins in replication.

Beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter.

beta-Casein gene expression in mammary epithelial cells is under the control of the lactogenic hormones, glucocorticoids, insulin, and prolactin. The hormonal control affects gene transcription, and several regulatory elements in the beta-casein gene promoter between positions -80 and -221 have previously been identified. A region located in the promoter between positions -170 and -221 contains overlapping sequences for negative and positive regulatory elements. A sequence-specific single-stranded DNA-binding factor (STR), composed of two proteins with molecular masses of 35 and 54 kDa, recognizes the upper strand of this region and has a repressing role in transcription. High-level STR binding activity was observed in nuclear extracts from mammary glands of pregnant and postlactating mice and from noninduced HC11 mammary epithelial cells, cells with a low level of transcriptional activity of the beta-casein gene. STR activity is downregulated in mammary epithelial cells during lactation of the animals and after lactogenic hormone induction of HC11 cells in culture. These cells strongly transcribe the beta-casein gene. We investigated the mechanism of downregulation and found that a lactogenic-hormone-induced molecule (I-STR) inhibits STR from binding to its DNA target. I-STR is composed of RNA. STR is sequestered into the cytoplasm by I-STR after lactogenic hormone induction of mammary epithelial cells and remains present in an RNA-bound form. A high-affinity STR binding site was found in the 5' untranslated region of beta-casein mRNA. We propose that beta-casein mRNA can function as I-STR. beta-Casein mRNA may positively regulate its own transcription by translocating STR from the nucleus to the cytoplasm. The beta-casein STR binding sequence increases expression of a transfected beta-galactosidase gene when it is placed into the 5' untranslated region sequence of the mRNA. STR may have a positive role in posttranscriptional regulation.

Beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter.

beta-Casein gene expression in mammary epithelial cells is under the control of the lactogenic hormones, glucocorticoids, insulin, and prolactin. The hormonal control affects gene transcription, and several regulatory elements in the beta-casein gene promoter between positions -80 and -221 have previously been identified. A region located in the promoter between positions -170 and -221 contains overlapping sequences for negative and positive regulatory elements. A sequence-specific single-stranded DNA-binding factor (STR), composed of two proteins with molecular masses of 35 and 54 kDa, recognizes the upper strand of this region and has a repressing role in transcription. High-level STR binding activity was observed in nuclear extracts from mammary glands of pregnant and postlactating mice and from noninduced HC11 mammary epithelial cells, cells with a low level of transcriptional activity of the beta-casein gene. STR activity is downregulated in mammary epithelial cells during lactation of the animals and after lactogenic hormone induction of HC11 cells in culture. These cells strongly transcribe the beta-casein gene. We investigated the mechanism of downregulation and found that a lactogenic-hormone-induced molecule (I-STR) inhibits STR from binding to its DNA target. I-STR is composed of RNA. STR is sequestered into the cytoplasm by I-STR after lactogenic hormone induction of mammary epithelial cells and remains present in an RNA-bound form. A high-affinity STR binding site was found in the 5' untranslated region of beta-casein mRNA. We propose that beta-casein mRNA can function as I-STR. beta-Casein mRNA may positively regulate its own transcription by translocating STR from the nucleus to the cytoplasm. The beta-casein STR binding sequence increases expression of a transfected beta-galactosidase gene when it is placed into the 5' untranslated region sequence of the mRNA. STR may have a positive role in posttranscriptional regulation.

Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.

The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.

Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.

The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.

Common double- and single-stranded DNA binding factor for a sterol regulatory element.

A cis-acting element necessary for sterol regulation, SRE-1, has previously been identified in the promoters of the low density lipoprotein receptor, hydroxymethylglutaryl (HMG)-CoA reductase, and HMG-CoA synthase genes. In this report we describe a nuclear factor, SRE-BF, isolated from Chinese hamster ovary nuclear extracts, that binds to the SRE-1 octanucleotide sequence. In addition to sequence-specific binding to SRE-1, as indicated by competition analysis with double-stranded DNA fragments, single-stranded oligomer DNA sequences also compete for binding in a sequence-specific fashion. Photochemical cross-linking experiments suggest that a common protein factor, with apparent molecular mass of 45-49 kDa, recognizes both single-stranded and double-stranded SRE-1. The binding specificity of SRE-BF to single-stranded SRE-1 closely correlates with the reported in vivo ability of SRE-1 to direct sterol responsiveness of transcription.