Wild-type p53 regulates human ribonucleotide reductase by protein-protein interaction with p53R2 as well as hRRM2 subunits.

Ribonucleotide reductase (RR) plays a key role in the synthesis of DNA and is the only enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides, providing a balanced supply of precursors for DNA synthesis and repair. There are three known human RR subunits, hRRM1, hRRM2, and p53R2, which is encoded by a p53 target gene. It is not clear whether p53 and RR can directly interact at the protein level to regulate DNA repair. It is also not known where deoxyribonucleotides are synthesized in the cell. In coimmunoprecipitation experiments, we found that hRRM2 and p53R2, but not hRRM1, bound to p53 in KB cells, which express wild-type p53. Moreover, in response to UV irradiation, both p53R2 and hRRM2 were released from p53 and shifted to bind hRRM1. Confocal microscopy confirmed the colocalization of p53 with p53R2 and hRRM2 and the translocation of hRRM1, p53R2 and hRRM2 from the cytoplasm to the nucleus after UV treatment. An in vivo RR activity assay showed that the kinetic profile of increased RR activity was consistent with the accumulation of RR subunits in the nucleus. The ability of p53R2 and hRRM2 to shift from binding p53 to hRRM1 in response to UV irradiation was deficient in the presence of mutant p53. Moreover, in cells overexpressing hRRM2, binding of p53R2 to p53 decreased, whereas binding to hRRM1 increased. Our results suggest that wild-type p53 directly interacts with both p53R2 and hRRM2. In response to UV irradiation, p53R2 and hRRM2 dissociate from p53 and p53R2, and hRRM2 and hRRM1 transfer to the nucleus and form an active RR complex to provide dNDPs for DNA repair. Therefore, the direct interaction of p53 with p53R2 and hRRM2 and the nuclear accumulation of RR subunits after UV exposure might play a pivotal role in DNA repair.

[1]  Yun Yen,et al.  Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential , 1998, Clinical & Experimental Metastasis.

[2]  J. G. Cory,et al.  Regulation of ribonucleotide reductase activity in mammalian cells , 2004, Molecular and Cellular Biochemistry.

[3]  Lijun Xue,et al.  The human ribonucleotide reductase subunit hRRM2 complements p53R2 in response to UV-induced DNA repair in cells with mutant p53. , 2003, Cancer research.

[4]  M. Oren,et al.  Regulation of p53 , 2002, Annals of the New York Academy of Sciences.

[5]  Y. Seo,et al.  p53 regulation of DNA excision repair pathways. , 2002, Mutagenesis.

[6]  J. Milner,et al.  The interaction of p53 with the nuclear matrix is mediated by F-actin and modulated by DNA damage , 2002, Oncogene.

[7]  N. Chang.  A potential role of p53 and WOX1 in mitochondrial apoptosis (review). , 2002, International journal of molecular medicine.

[8]  L. Feng,et al.  Interaction of p53 and DNA-PK in response to nucleoside analogues: potential role as a sensor complex for DNA damage. , 2001, Cancer research.

[9]  Y. Nakamura,et al.  p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint. , 2001, Cancer research.

[10]  Yusuke Nakamura,et al.  Mammalian p53R2 Protein Forms an Active Ribonucleotide Reductasein Vitro with the R1 Protein, Which Is Expressed Both in Resting Cells in Response to DNA Damage and in Proliferating Cells* , 2001, The Journal of Biological Chemistry.

[11]  C. Prives,et al.  p53 accumulates but is functionally impaired when DNA synthesis is blocked. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  K. Vousden,et al.  A ribonucleotide reductase gene is a transcriptional target of p53 and p73 , 2000, Oncogene.

[13]  A. Chabes,et al.  Controlled Protein Degradation Regulates Ribonucleotide Reductase Activity in Proliferating Mammalian Cells during the Normal Cell Cycle and in Response to DNA Damage and Replication Blocks* , 2000, The Journal of Biological Chemistry.

[14]  S. Elledge,et al.  Cancer: p53 sends nucleotides to repair DNA , 2000, Nature.

[15]  Yusuke Nakamura,et al.  A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage , 2000, Nature.

[16]  P. May,et al.  Twenty years of p53 research: structural and functional aspects of the p53 protein , 1999, Oncogene.

[17]  Margaret Ashcroft,et al.  Regulation of p53 stability , 1999, Oncogene.

[18]  Y. Yen,et al.  Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. , 1999, Cancer research.

[19]  V. Rotter,et al.  Introduction: p53 – the first twenty years , 1999, Cellular and Molecular Life Sciences CMLS.

[20]  T. Kinsella,et al.  Expression of ribonucleotide reductase after ionizing radiation in human cervical carcinoma cells. , 1998, Cancer research.

[21]  P. Reichard,et al.  Ribonucleotide reductases. , 1998, Annual review of biochemistry.

[22]  W. El-Deiry,et al.  Regulation of p53 downstream genes. , 1998, Seminars in cancer biology.

[23]  A. Fornace,et al.  p53-mediated protective responses to UV irradiation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Hanawalt,et al.  Expression of Wild-type p53 Is Required for Efficient Global Genomic Nucleotide Excision Repair in UV-irradiated Human Fibroblasts* , 1997, The Journal of Biological Chemistry.

[25]  A. Levine.  p53, the Cellular Gatekeeper for Growth and Division , 1997, Cell.

[26]  K. Vousden,et al.  p53 in signaling checkpoint arrest or apoptosis. , 1996, Current opinion in genetics & development.

[27]  P. O'Connor,et al.  Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. , 1995, Oncogene.

[28]  E. Johansson,et al.  Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Fornace,et al.  Induction of Cellular p53 Activity by DNA-Damaging Agents and Growth Arrest , 1993, Molecular and cellular biology.

[30]  Reddy Gp,et al.  Replitase: a complex integrating dNTP synthesis and DNA replication. , 1993 .

[31]  R. Fager,et al.  Replitase: a complex integrating dNTP synthesis and DNA replication. , 1993, Critical reviews in eukaryotic gene expression.

[32]  L. Sun,et al.  Escherichia coli ribonucleotide reductase expression is cell cycle regulated. , 1992, Molecular biology of the cell.

[33]  J. Wright,et al.  Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent. , 1992, The Journal of biological chemistry.

[34]  A. Milosavljevic,et al.  Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[35]  W. Isaacs,et al.  Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. , 1991, Cancer research.

[36]  A. K. Chan,et al.  Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. , 1990, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[37]  E. Nodzenski,et al.  Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild‐type and mutant S49 T lymphoma cells , 1990, Journal of cellular physiology.

[38]  R. W. Davis,et al.  DNA damage induction of ribonucleotide reductase , 1989, Molecular and cellular biology.

[39]  G. Mann,et al.  Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation. , 1988, Cancer research.

[40]  Y. Engström,et al.  Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase. , 1988, The EMBO journal.

[41]  C. Mathews,et al.  Eukaryotic DNA metabolism. Are deoxyribonucleotides channeled to replication sites? , 1986, Experimental cell research.

[42]  Y. Engström,et al.  Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits. , 1985, The Journal of biological chemistry.

[43]  S. Eriksson,et al.  Cell cycle-dependent regulation of mammalian ribonucleotide reductase. The S phase-correlated increase in subunit M2 is regulated by de novo protein synthesis. , 1984, The Journal of biological chemistry.

[44]  J. G. Cory,et al.  Studies directed toward testing the "channeling" hypothesis--ribonucleotides----DNA in leukemia L1210 cells. , 1984, Biochemical and biophysical research communications.

[45]  A. Mozo-Villarías.  Fluorescence study of the effects of histone tyrosyl residues of DNA. , 1984, Biochemical and biophysical research communications.

[46]  H. Hansson,et al.  Localization of ribonucleotide reductase in mammalian cells. , 1984, The EMBO journal.

[47]  A. Pardee,et al.  Multienzyme complex for metabolic channeling in mammalian DNA replication. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[48]  G. Bjursell,et al.  Nuclear and cytoplasmic pools of deoxyribonucleoside triphosphates in Chinese hamster ovary cells. , 1974, The Journal of biological chemistry.