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Carolina Ochoa

Zhang, J., H. Willers, Z. Feng, J.C. Ghosh, S. Kim, D.T. Weaver, J.H. Chung, S.N. Powell, and F. Xia. 2004. Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair. Molecular and cell biology 24: 708-718. . Carolina Ochoa. Outline. Cell cycle and checkpoints p53

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Carolina Ochoa

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  1. Zhang, J., H. Willers, Z. Feng, J.C. Ghosh, S. Kim, D.T. Weaver, J.H. Chung, S.N. Powell, and F. Xia. 2004.Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair.Molecular and cell biology 24: 708-718. Carolina Ochoa

  2. Outline • Cell cycle and checkpoints • p53 • Ataxia telangiectasia mutated (ATM) • Chemokine 2 (Chk2) • Breast cancer gene suppressor 1 (BRCA1) • DNA double-strand break (DSBs) • Study

  3. It is governed by a genetic program The disruption of the regulation can lead to uncontrolled cell division Many genes are involved At least three major checkpoint exist At a checkpoint the cell is “checked” before it can continue the cycle The Cell Cycle

  4. The genes that regulate checkpoints are enzymes called cdc (cell division cycle mutation) kinases that can add phosphates to other proteins

  5. The Cell Cycle Checkpoints • G1/S checkpoint monitors cell size and DNA integrity • G2/M checkpoint monitors completion of DNA synthesis and DNA damage • M/G1 checkpoint monitors spindle formation and their attachments to kinetochores

  6. The Cell Cycle Checkpoints Fig.1 Hoffman et al. 2005

  7. p53 • Protein product involved in scrutiny of the cell cycle at G1/S checkpoint • If G1/S checkpoint detects DNA DSBs and if the normal gene is present the cell is programmed for apoptosis (cell death) • If both copies of the gene are mutated, abnormal functioning occurs, the cell may continue uncontrollable cell growth

  8. ATM and Chk2 • ATM is a protein that: • detects DNA damage (especially DSBs) • interrupts with p53 the cell cycle when there is damage • Chk2 is a kinase

  9. BRCA1 • A gene located on chromosome 17 • It is necessary for cell cycle checkpoint, DNA repair, regulation of transcription, apoptosis, and others. • Dysfunction causes cancer development

  10. DNA DSBs • Very dangerous form of DNA damage • Two ways to repair: 1. Homologous recombination (HR) - requires undamaged molecules that contain homologous DNA sequence mediated through multiple proteins 2. Non-homologous recombination (NHR) - an error-prone process in which nucleotide alterations are tolerated at the sites of rejoining

  11. Chk2 phosporylation of BRCA1 regulates DNA double-strand break repair Zhang et al.

  12. Study • Discover the roles of Chk2- and ATM-mediated BRCA1 phosphorylation in the regulation of HR and NHR • To do this the Rad51- dependent HR and MRN-dependent NHR were characterized in a defined human cancer cell line

  13. Chromosomal HR in breast cancer cells Fig. 2.

  14. HR, radiation sensitivity, and S-phase checkpoint in BRCA1 Fig. 3.

  15. What is the relationship between Rad51 and BRCA1? 10% 66.8% 60.5% 32.1% 27.8% 10% 10 % 10 % Fig. 4.

  16. Chromosomal integration in NBS fibroblasts and parental cells Fig. 5.

  17. What is the relationship between Mre11 and BRCA1? 71.9 % 77.9% 35.1% 44.5% 6% 6% 6% 6% Fig. 6.

  18. What is the influence of Chk2 kinase activity on NHR? Fig. 7A

  19. What is the influence of Chk2 kinase activity on HR? Fig. 7B

  20. What is the Rad 51 percentage of wt Chk2 and kd Chk2? Fig. 7C

  21. Results • The promotion of HR and suppression of NHR are governed by the serine 988 residue of BRCA1 • Chromosomal HR is facilitated by BRCA1 (Fig. 3) • Chk2 protein kinase activity on BRCA1 is essential in dictating the regulation of both HR and NHR (Fig. 3B, 7B and C). • ATM-mediated phosphorylation was not required for the function of BRCA1 in regulation of either HR or NHR (Fig. 3-6). • The relationship between BRCA1 and NHR is an active effect of BRCA1.

  22. Discussion • The promotion of HR mediated by the Chk2-BRCA1 pathway may contribute to the prevention of breast cancer development. • HR may play a significant role in maintaining chromosome stability. • BRCA1 may exert regulatory control of HR and NHR at an early stage. • BRCA1 may be involved in a dual regulatory model (Fig. 8)

  23. What is the role of BRCA1 in recombinational repair? Fig.8.

  24. Literature Cited • Hoffman I., K. Khanna, M. O’Connel, N. Walworth, and T.J. Yen. 2005. Cell cycle checkpoints. Reactome org. <http://www.reactome.org/cgi-bin/eventbrowser?DB=gk_ currents&ID=69620&> • Klug W.S., and M.R. Cummings. 2005. Essential of genetics, fifth edition. Pearson prentice hall: 26-27 and 497. • Zhang, J., H. Willers, Z. Feng, J.C. Ghosh, S. Kim, D.T. Weaver, J.H. Chung, S.N. Powell, and F. Xia. 2004. Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair. Molecular and cell biology 24: 708-718. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmedid=14701743. • Poznak C.V. and A.D. Seidman. 2002. Breast cancer. Encyclopedia of breast cancer, second edition. Elsevier science: 287-299.

  25. Questions?

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