Abstract
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.
Highlights
There is a close correlation between mammalian ribonucleotide reductase activity and the rate of DNA synthesis with no
It is reasonable to assume that ribonucleotide reductase may be involved in the repair, at least in nonproliferating cells, since deoxyribonucleotides are present in very low levels in cells outside S-phase [1, 2]
Hydroxyurea treatment without irradiation did not affect the amount of single-stranded DNA. This indicates that in the presence of hydroxyurea there is an accumulation of excised gaps in DNA that cannot be filled in with newly synthesized DNA most probably due to lack of deoxyribonucleotides
Summary
Cell Culture, Synchronization, Transfection, and Selection of Stable Transformants—Balb/3T3 cells (ATCC CCL 163) and hydroxyurea-resistant, R2-overproducing mouse TA 3 cells were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated horse serum as described earlier [5]. After h, cells were washed with phosphate-buffered saline (PBS); ml of medium containing 0.6% fetal calf serum were added; and the cells were incubated for another 48 h. The plasmid p19lucR2 TTTAAA contains only the TTTAAA box and the transcription start from the R2 promoter (nt Ϫ47 to ϩ17) ligated to the luciferase reporter gene [12]. In the case of methyl methanesulfonate treatment, the cells were washed as described above and incubated for 30 min at 37 °C in the presence of prewarmed PBS containing the drug at 2.36 –118 M concentrations. After serum starvation and treatment with UV light, DNA single strand breaks were measured by the method developed by Erixon and Ahnstrom [23]. The RNA was denatured with glyoxal, electrophoresed on a 1% agarose gel, blotted to a nitrocellulose filter, and hybridized to a full-length R2 cDNA probe as described [5, 25]
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