Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide ؊672 to nucleotide ؊527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.Ribonucleotide reductase (EC 1.17.4.1) is a key enzyme in DNA precursor synthesis reducing all four ribonucleotides to the corresponding deoxyribonucleotides (1, 2). Mouse ribonucleotide reductase is a heterodimer composed of the two homodimeric subunits, proteins R1 and R2, each inactive alone. Enzyme activity is cell cycle regulated with low or undetectable levels in G 0 /G 1 and maximal activity in the S phase of the cell cycle (3).The mouse R1 and R2 mRNA expression is S phase specific with very low or undetectable levels in G 0 /G 1 cells, a pronounced increase as cells progress into S phase, and a decline when cells progress into G 2 ϩ M (4). Reporter gene constructs show that the R2 promoter is activated almost immediately after quiescent G 0 /G 1 synchronized cells are released by serum readdition. Promoter activity then increases steadily, reaching its maximum at around 12 h after serum readdition (5). From the early promoter activation, the R2 gene could be classified as an immediate early response gene. However, in vitro studies demonstrated that this early activation only results in the synthesis of immature short R2 mRNA transcripts due to a G 1 -specific transcriptional block located in the first intron of the R2 gene (5). This block is not released until cells reach S phase when full-length transcripts are synthesized. Reporter gene constructs containing the R2 promoter-1st exon/1st intron indicate that the transcriptional block is active also in vivo, and S phase-specific protein binding was identified to a DNA region just upstream from the block. The mouse R2 promoter contains a TTTAAA motif at position nt 2 Ϫ24 and a CCAAT motif at position nt Ϫ75 upstream from the transcription start (6). DNase I footprinting analyses revealed four DNA-protein binding regions within the R2 promoter. The region most proximal to the transcription start (nt Ϫ93 to Ϫ56) includes the CCAAT box and is called ␣. The other three DNA-protein binding re...

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides required for DNA synthesis and consists of two nonidentical subunits, proteins R1 and R2. Here we show that the R1 promoter can be induced up to 3-fold, and the R2 promoter is induced up to 10-fold by UV light in a dose-dependent manner. This was demonstrated using serum-starved, synchronized G0/G1 mouse fibroblast 3T3 cells stably transformed with different R1 and R2 promoter-luciferase reporter gene constructs. R2 promoter activation requires a minimal promoter, containing a TTTAAA element plus the transcription start, and either three upstream DNA-protein binding regions or one proximal, NF-Y binding region. This is different from proliferation-specific activation of the R2 promoter. Using Northern blotting we show a preferential accumulation of the minor, 1. 6-kilobase R2 transcript in irradiated cells, whereas the levels of the major 2.1-kilobase transcript are unchanged. No R2 promoter activation was observed after treatment of mouse cells with agents reported to induce the ribonucleotide reductase genes in Saccharomyces cerevisiae such as hydroxyurea or methylmethane sulfonate. This indicates that activation of ribonucleotide reductase gene expression is specific for nucleotide excision repair in mammalian cells and not part of a general response to DNA damage.

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903Ribonucleotide reductase (EC 1.17.4.1 -2) catalyses the direct reduction of ribonucleotides to the corresponding deoxyribonucleotides. This reaction enabled the evolution from the early 'RNA world' to the present 'DNA world'. Storage of genetic information as DNA instead of RNA is much more stable and this was a prerequisite for the later development of multiple, separate, species [l].Ribonucleotide reduction is a chemically difficult reaction and requires free radical chemistry. We now know of three different classes of ribonucleotide reductases [ 11. Catalysis in all three classes is dependent on an enzyme-centred free radical, which in the class I enzymes is an iron-centre-generated tyrosyl free radical. Class I1 uses adenosyl cobalamin as a cofactor, which generates a transient radical during catalysis. Finally, class I11 uses a glycine radical which is generated in a reaction that requires S-adenosylmethionine.All plant and eukaryotic ribonucleotide reductases described so far belong to class I. Here the enzyme consists of two non-identical homodimeric subunits, proteins R1 and R2. This is very similar to the extensively studied Escherichiu coli nrd AB enzyme [2-41. The 2 x 90 kDa mouse R1 protein contains the ribonucleoside diphosphate substrate binding sites as well as two different types of nucleoside triphosphate effector binding sites. It also contains a number of redox active disulphides that participate in electron transfer from NADPH/thioredoxin/ glutaredoxin. There is only one enzyme catalysing the reduction of all four different ribonucleotides, but both the overall activity and the substrate specificity are controlled by allosteric regulation. This control ensures a balanced supply of the four deoxyribonucleotides during DNA replication and repair to prevent misincorporation and mutagenesis [3,5].Each 45 kDa polypeptide of the mouse R2 protein contains a binuclear iron centre which, during its formation, generates a stable tyrosyl free radical that is essential for activity [6,7]. The iron centre was shown to be labile at physiological temperatures, resulting in a loss of about *To whom correspondence should be addressed. 50% of the iron after 30 min at 37°C [8]. Therefore there is a continuous regeneration of the iron-tyrosyl radical centre in mammalian R2 in vivo. The regeneration requires ferrous iron and oxygen and explains why mammalian ribonucleotide reductase activity, and consequently also DNA synthesis and cell growth, is sensitive to iron chelators and oxygen deprivation. This was demonstrated in direct EPR measurements of the R2 tyrosyl radical in intact cells treated with iron chelators [8]. The lability of the iron centre in the mammalian R2 protein is in marked contrast to the E. coli R2 protein, which does not easily lose its iron.The crystal structures of the E. coli R1 and R2 proteins show that the tyrosyl free radical is buried lOA (1 nm) away from the surface of the very rigid R2 protein [9]. Furthermore, the active site of the R1 protein is located 25A from the most likely R...

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