STRUCTURE AND PROMOTER CHARACTERIZATION OF THE GENE ENCODING THE LARGE SUBUNIT (R1-PROTEIN) OF MOUSE RIBONUCLEOTIDE REDUCTASE

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two no nidentical subunits, proteins R1 and R2, both required for enzyme acti vity. The structure of the genomic mouse ribonucleotide reductase R1 g ene was compiled from a number of overlapping lambda clones isolated f rom a Charon 4A mouse sperm genomic library. The R1-encoding gene cove rs 26 kb and consists of 19 exons. All exon-intron boundaries were loc ated by dideoxynudeotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flankin g region of the R1-encoding gene were cloned and sequenced, and the tr anscriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially ident ified by using gel-retardation mobility-shift assays and UV crosslinki ng. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-d ependent R1 gene expression.