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Ribonucleotide reductases provide the building blocks for DNA replication in all living cells. Three different classes of enzymes use protein free radicals to activate the substrate. Aerobic class I enzymes generate a tyrosyl radical with an iron-oxygen center and dioxygen, class II enzymes employ adenosylcobalamin, and the anaerobic class III enzymes generate a glycyl radical from S-adenosylmethionine and an iron-sulfur cluster. The X-ray structure of the class I Escherichia coli enzyme, including forms that bind substrate and allosteric effectors, confirms previous models of catalytic and allosteric mechanisms. This structure suggests considerable mobility of the protein during catalysis and, together with experiments involving site-directed mutants, suggests a mechanism for radical transfer from one subunit to the other. Despite large differences between the classes, common catalytic and allosteric mechanisms, as well as retention of critical residues in the protein sequence, suggest a similar tertiary structure and a common origin during evolution. One puzzling aspect is that some organisms contain the genes for several different reductases.

Because of the heterogeneity of chromatin, the site of integration of human immunodeficiency virus (HIV) in the genome could have dramatic effects on its transcriptional activity. We have used an HIV‐1‐derived retroviral vector, in which the green fluorescent protein is under the control of the HIV promoter, to generate by infection 34 Jurkat clonal cell lines each containing a single integration of the HIV‐1 vector. In the absence of Tat, a 75‐fold difference in expression level between the highest and lowest expressing clones was observed. Basal promoter activity was low in 80% of the clones and moderate to high in the remaining 20% of clones. We found that differences in expression levels are due to the integration site and are not controlled by DNA methylation or histone acetylation. Tat activated transcription in each clone, and an inverse correlation was observed between basal transcriptional activity and inducibility by Tat. These observations demonstrate that the chromatin environment influences basal HIV gene expression and that the HIV Tat protein activates transcription independently of the chromatin environment.

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants. Eukaryotic DNA is packaged into chromatin through its association with histone proteins. The linker histone H1 sits at the base of the nucleosome near the DNA entry and exit sites to stabilize two full turns of DNA. In particular, histone H1 participates in nucleosome spacing and formation of the higher-order chromatin structure. In addition, H1 seems to be actively involved in the regulation of gene expression. Histone H1 in mammals is a family of closely related, single-gene encoded proteins, including five somatic subtypes (from H1.1 to H1.5) and a terminally differentiated expressed isoform (H1.0). It is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible knock-down of each of the H1 variants in breast cancer cells. A different subset of genes is altered in each H1 knock-down, and depletion has different effects on cell survival. Interestingly, H1.2 and H1.4 depletion specifically caused arrest of cell proliferation. Concomitant with this, H1.2 depletion caused decreased global nucleosome spacing and repressed expression of a number of cell cycle genes. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants.