Insect Molecular Biology (2004) 13(3), 231–239 © 2004 The Royal Entomological Society 231 Blackwell Publishing, Ltd. The mosquito ribonucleotide reductase R2 gene: ultraviolet light induces expression of a novel R2 variant with an internal amino acid deletion G. Jayachandran* and A. M. Fallon Department of Entomology, University of Minnesota, St. Paul, MN, USA Abstract Using RT-PCR, we examined expression of the ribonu- cleotide reductase R2 subunit (RNR-R2) in Aedes albopictus mosquito cells after treatment with ultra- violet light (UV). In control cells, a predominant band at 1.2 kb corresponded to the full-length cDNA. A smaller 650 bp band was unique to UV-treated cells. Sequence analysis showed that the 650 bp band encoded a pro- tein with an internal deletion of 179 amino acids, rela- tive to Ae. albopictus RNR-R2. The N-terminal twenty amino acids were identical between AalRNR-R2 and Aal∆ R2; downstream of the deletion, the proteins differed at only four residues. In Aal ∆ R2, the internal deletion spanned five residues critical to RNR-R2 enzymatic activity, including a key tyrosine residue that generates an essential free radical. The full-length 46 kDa and truncated 25 kDa RNR-R2 proteins were shown to be expressed on Western blots, and to differ in their subcellular localization. Similarly, expression of the two proteins was differentially regulated during the cell cycle, and expression of Aal ∆ R2 predominated after UV treatment. Aal∆ R2 resembled a human RNR- R2 variant called p53R2, which was induced by agents that damage DNA. As was the case with p53R2 and its antisense RNA, levels of Aal ∆ R2 were diminished after treatment of mosquito cells with RNAi corresponding to p53 from Drosophila melanogaster . Examination of the AalRNR-R2 homologue in the Anopheles gambiae genome suggested that Aal ∆ R2 resulted from precise splicing between Exons 1, 4 and 5, eliminating Exons 2 and 3. The likelihood that Aal ∆ R2 is a non-enzymatic, functional participant in DNA metabolism is suggested by enhancement of DNA repair in an in vitro system and by the presence of a similar gene ( rnr4 ) in yeast. Keywords: mosquito cell line, cell cycle, DNA repair, ultraviolet light, Anopheles gambiae genome. Introduction Ribonucleotide reductase (RNR) converts ribonucleotides to the corresponding deoxyribonucleotides (dNTPs) and maintains the relative amounts of the dNTPs that constitute the precursor pool for de novo DNA synthesis (Thelander & Reichard, 1979; Jordan & Reichard, 1998). In eukaryotic cells, the active enzyme is a tetramer composed of two R1 homodimers and two R2 homodimers. The ∼ 170 kDa RNR- R1 homodimer contains both the active site and the binding sites for allosteric effectors, whereas the ∼ 88 kDa RNR-R2 homodimer contains the binuclear iron centre, which is involved in formation of a tyrosyl free-radical required for catalytic activity (reviewed by Jordan & Reichard, 1998). In mammals, the mRNAs encoding RNR-R1 and RNR- R2 are cell cycle dependent, and their transcription increases sharply in the S-phase (Engstrom et al ., 1985). The abundance of R1 protein is maintained at a constant level and in excess of R2, whereas translation of R2 mRNA into protein is S-phase specific (Eriksson et al ., 1984), and the protein has a half-life of only 3 h (Chabes & Thelander, 2000). Thus R2 abundance regulates the overall rate of RNR activity, and changes in R2 protein abundance may provide a safety mechanism that would ensure an adequate supply of dNTPs for replication and repair during S and G2.The rapid degradation of R2 protein in late mitosis and its absence in G0/G1 cells hence prevent re-replication of the genome (Chabes & Thelander, 2000). Regulation of RNR activity by R2 abundance fails to account for the observation that DNA repair occurs in G1 cells and in nonproliferating cells that lack detectable R2 protein. During an attempt to identify target genes for the tumour suppressor protein, p53, Tanaka et al . (2000) identified a novel peptide similar to human R2, which was induced by agents that damage DNA, such as ultraviolet light (UV), γ -irradiation and adriamycin. More recently, Guittet et al . (2001) showed that, in mouse and human cells, this Received 30 July 2003; accepted after revision 8 December 2003. Corre- spondence: Dr Ann M. Fallon, Department of Entomology, 1980 Folwell Ave., St. Paul, MN 55108, USA. Tel.: +1 612 625 3728; fax: +1 612 625 5299; e-mail: fallo002@tc.umn.edu *Present address: Department of Thoracic and Cardiovascular Surgery, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.