Electron microscopy shows that complexes of the single-strand DNA 60, 105, 118, 130, 145, 150, and 210 bases. Isopycnic banding in CsCl solution yields densities of 1.272 and 1.700 g/ml, respectively, for SSB alone and for fd DNA and, after fixation, of 1.388 g/ml for fd DNA-SSB beaded fibers and 1.373 g/ml for the individual protein-DNA beads. Based on these data and the molecular weights of SSB and fd DNA, we suggest that the nucleoprotein chain consists of eight molecules of SSB bound to 145 bases of DNA, with these units linked by roughly 30 bases of protein-free DNA. The excellent concord between results obtained by enzyme digestion of unfixed native samples and, after fixation, by electron microscopy and density banding supports the conclusion that SSB organizes single-stranded DNA in a manner similar to the organization of duplex DNA by histones.DNA binding proteins perform vital roles in all cells. A singlestranded DNA (SS DNA) binding protein present in Escherichia coli (SSB) was first identified and purified by Sigal et al.(1). Isolation ofmutants of this protein and in vivo studies have shown that it is required in replication of the chromosomes of E. coli and several small DNA phages (2-5) and involved in genetic recombination and SOS repair (6-9). One must conclude that SSB interacts in a precise manner with enzymes in the replication and recombination pathways and that changes in these interactions are highly detrimental to the cell. Despite the central role of this protein, little is known about the threedimensional structure of the SSB-DNA complex. This represents a serious gap in our knowledge because it is likely that the structure of the SSB-DNA complex is a key element in its interaction with other proteins, in particular with RecA, another SS DNA binding protein, also present in substantial amounts in the normal cell.In previous studies, electron microscopy (EM) has been used to visualize SSB-SS DNA complexes (10, 11) and a regular beaded substructure has been noted (12). Although it has been shown that the fixation methods used in those studies retain the native features ofnucleoprotein structures (13,14), in any morphological study by microscopy, the structural features of apparent interest may reflect the preparative procedure and not the native form. (18). Isolation of phage fd SS DNA labeled with 32p (ICN) was as described by Makino et al (19). SSB-fd SS DNA complexes were formed at a protein/ DNA ratio of 10: 1 unless otherwise stated.Enzymes and Reagents. DNase I (Sigma) was further purified by affinity chromatography on Agarose-UMP, as described by Brison and Chambon (20). Micrococcal nuclease was from Worthington.Gel Electrophoresis. Products of DNase I digestions were analyzed on 10% polyacrylamide gels as described by Peacock and Dingman (21). Electrophoresis of micrococcal nuclease digestion products was on 4-10% polyacrylamide gels with an acrylamide/bis-acrylamide ratio of 30:1.2. The gels and running buffer were 0.02 M Tris base/0.04 M NaOAc/1 mM EDTA. All gels...

Cell cycle-dependent histone genes are transcribed at a basal level throughout the cell cycle, with a three- to fivefold increase during early S phase. Protein-DNA interactions in the 5' promoter region of a cell cycle-regulated human H4 histone gene have been analyzed at single-nucleotide resolution in vivo. This region contains two sites, with four potential protein-binding domains, at which the DNA is protected from reaction with dimethyl sulfate in cells and from digestion with deoxyribonuclease I in nuclei. These protein-DNA interactions persist during all phases of the cell cycle and dissociate with 0.16 to 0.2M sodium chloride.

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Epidermal growth factor receptor (EGFR) is a transmembrane receptor whose overexpression in breast cancer predicts for poor prognosis and is inversely correlated with expression of estrogen receptor (ER). This study was designed to investigate whether estrogen plays an active role in suppression of EGFR expression in estrogen-responsive breast cancer cell lines expressing low levels of EGFR. Upon withdrawal of estrogen, EGFR mRNA and protein increased 3±6 fold in MCF-7, T47D, and BT474 ER breast cancer cells. This was reversible upon addition of estradiol back to the culture media, but only after prolonged treatment. Nuclear run-on assays and studies with the transcription inhibitor actinomycin D demonstrated that regulation is at the transcriptional level. These results indicate that in the presence of estrogen, ER breast cancer cells possess active mechanisms to suppress EGFR expression. Up-regulation of EGFR in response to estrogen depletion and growth inhibition could represent an attempt to rescue cell growth by utilizing an alternative pathway. Indeed, we found that estrogen-depleted breast cancer cells are more sensitive to the mitogenic effects of EGF and TGF-a, and simultaneous blockade of both estrogen and EGFR signaling pathways induced cell death.