Sequence-specific single stranded DNA binding

Abstract: Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

Abstract: The promoter region of the chicken alpha2(I) collagen gene contains a pyrimidine-rich element that is well conserved in different mammalian species. This sequence can also form an unusual DNA structure as shown by its sensitivity to SI nuclease in vitro and it lies in a region that is DNase I-hypersensitive only when this promoter is active. We have recently reported that fibroblast nuclear proteins, including chicken Y-box-binding protein 1, bind to this single-stranded pyrimidine-rich sequence. Here we report the isolation, from a chick embryo fibroblast cDNA expression library, of a partial cDNA clone encoding a previously unknown protein, designated SSDP (sequence-specific single-stranded DNA-binding protein), that binds this single-stranded sequence. This clone contains 1199 bp of chicken sequence and has a single long open reading frame that encodes 284 amino acid residues. The affinity-purified recombinant protein encoded by this cDNA binds sequence-specifically to the single-stranded pyrimidine sequence. This cDNA sequence lacks significant similarity to any known gene in the data banks, but it is highly conserved in expressed sequence tags derived from both mouse and human. The corresponding amino acid sequence is remarkably conserved, having 97% identity with mouse and human expressed sequences. The corresponding mRNA is approx. 1800 nt in length and is expressed in both fibroblasts and chondrocytes. The high affinity of this protein for this conserved pyrimidine-rich region suggests that it might be involved in the transcriptional regulation of the alpha2(I) collagen gene.

Abstract: Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes.