Ribonuclease H (hybrid) in Escherichia coli. Identification and characterization.
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Biomedical subjects
Publications and source records attributed to A D Riggs.
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Binding of lac repressor to 20 synthetic DNAs of high molecular weight with defined repeating sequences was investigated by competition experiments. Although none of these DNAs binds repressor as tightly as does lac operator, most do bind to a measurable extent. Their affinity for repressor varies greatly and is a function of both nucleotide composition and sequence. Poly(dC-dC).poly(dG-dC) competes for repressor 200-times less well than either poly(dA-dT).poly(dA-dT) or poly(dT-dT-dG).poly(dC-dA-dA). The other DNAs show a broad spectrum of affinities for repressor between these extremes. These results show that the lac repressor has affinity for, and can distinguish between, sequences distantly related to its operator.
As measured by a decreased rate of dissociation, lac repressor binds 10-times tighter to 5-bromodeoxyuridine-substituted lac operator than it does to normal lac operator. This result is obtained both in the absence and in the presence of isopropylthiogalactoside, an inducing ligand. These data are significant with regard to the mechanism of sequence-specific protein-DNA interaction, and also suggest a possible explanation for the effects of bromodeoxyuridine on the expression of differentiated functions in eukaryotic cells.
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A protein required for the activation of the lac operon has been extensively purified and partly characterized. This protein, called CGA protein (catabolite gene activator protein, sometimes named CAP), is a dimer with subunits of 22,000 daltons. Purified CGA protein has a substantial affinity for DNA; this affinity is greatly strengthened by cAMP and strongly inhibited by cGMP. Other studies have shown that these cyclic nucleotides compete for a binding site on CGA protein. The opposing effects of the two cyclic compounds in DNA-CGA protein binding show a parallel behavior to their effects on the expression of the lac operon. Thus cAMP, in addition to CGA protein, is required for expression of the lac operon, whereas cGMP inhibits the expression. The obvious inference is that CGA protein activates the lac operon by binding to the DNA under the influence of cAMP. Thus, CGA protein seems to be a new type of regulatory protein: a DNA-binding activator.
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In this paper we address the question: How do replicating mammalian cells remember with high fidelity their proper state of differentiation? Several possible mechanisms for cell memory are discussed, and it is concluded that only mechanisms involving DNA methylation are supported by strong experimental evidence. This evidence is reviewed. The establishment and modulation of methylation patterns are discussed and a hemimethylation model for stem cells is presented. The overall conclusion is that, although little is yet known about the details, there should be little doubt about the existence of a methylation system functioning at least to aid cell memory.