In eukaryotic cells, DNA damage-induced by UV radiation activates checkpoints that delay cell cycle progression. In the fission yeast S. pombe, numerous genes are involved in response to DNA damage including rad1, whose activation results in G2 phase arrest. To identify functional homologs in metazoans, we used a Xenopus cDNA library expressed in S. pombe to screen for restoration of UV resistance to rad1-deficient cells. We identified a novel Xenopus gene, designated Speedy (or Spy1), whose expression partially restores the rad1 DNA damage checkpoint. Microinjection of Speedy mRNA into Xenopus oocytes was able to induce rapid meiotic maturation in the absence of progesterone, the normal inducer of meiotic maturation. Speedy-induced maturation proceeds through activation of MAPK and M-phase-promoting-factor (MPF; the universal M-phase regulator that controls meiotic progression), a complex of cyclin B and cdc2. Furthermore, Spy1 was found to bind directly to and prematurely activate cdk2.

We isolated the human homolog of Speedy by PCR-based cloning from a human testis cDNA library. This gene encodes for a polypeptide of 286 residues and shares an overall homology of approximately 40% with Xenopus Speedy. Microinjection of HuSpy1 mRNA into Xenopus oocytes is able to induce maturation in the absence of hormone. Immunoprecipitation from transfected 293T cells reveals that Myc-HuSpy1 directly binds to cdk2, as does Xenopus Speedy. Furthermore, transient transfection of HuSpy1 into 293T cells increases the rate of proliferation for these cells. FACS analysis confirms thatHuSpy1 expression reduces the amount of cells in G1 and increases the number of cells in S and G2/M. This result indicates that HuSpy1 is a novel mammalian cell cycle regulator, and, coupled with preliminary data that HuSpy1 can bypass a nocodazole-induced spindle checkpoint, we are currently investigating the role of Spy1 in cell cycle checkpoint control. INSERTPARA2

PUBLICATIONS (resulting from this training, and some recent ones)

Lenormand JL, Dellinger RW, Knudsen KE, Subramani S, Donoghue DJ. (1999) EMBO J. 18:1869-1877. Speedy: a novel cell-cycle regulator of the G2/M transition. EMBO J. 18:1869-1877.

Porter, LA, Dellinger RW, Tynan JA, Barnes EA, Kong M, Lenormand JL, Donoghue DJ. (2002) Cell Biol. 157:357-366. Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of cdk2. J. Cell Biol. 157:357-366.

Barnes, EA, Porter LA, Lenormand JL, Dellinger RW, Donoghue DJ. (2003) Human Spy1 promotes survival of mammalian cells following DNA damage. Cancer Research 63:3701-3707.

Dellinger RW, Karjian PL, Neuteboom ST. (2003) NB1011 induces Ser15 phosphorylation of p53 and activates the G2/M checkpoint. Anticancer Drugs. 14:449-55.

Dellinger RW, Fang JL, Chen G, Weinberg R, Lazarus P. (2006) Importance of UDP-glucuronosyltransferase 1A10 (UGT1A10) in the detoxification of polycyclic aromatic hydrocarbons: decreased glucuronidative activity of the UGT1A10139Lys isoform. Drug Metab Dispos. 34:943-9.

Sun D, Chen G, Dellinger RW, Duncan K, Fang JL, Lazarus P. (2006) Characterization of tamoxifen and 4-hydroxytamoxifen glucuronidation by human UGT1A4 variants. Breast Cancer Res. 8:R50.