BRCA1 Maintains Genomic Integrity by Regulating DNA Repair via Double Strand Break Repair Methods in breast cancer cell

1. BRCA1 Maintains Genomic Integrity by Regulating DNA Repair via Double Strand Break Repair Methods in breast cancer cells Carolina Ochoa Seminar

2. The cell cycle • It is governed by a genetic program because of it is genetically regulated • The disruption of the regulation can lead to uncontrolled cell division that leads to malignant tumors such as breast cancer • Uncontrolled cell division can occur only by a failure of checkpoint G1/S or G2/M • Many genes are involved in the cell cycle • At least three major checkpoint exist • At a checkpoint the cell is “checked” before it can continue the cycle

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

4. The cell cycle checkpoints • G1/S checkpoint monitors cell size and DNA integrity • If it fails, the cell cycle is interrupted until the problem is corrected • If both are normal, the cell cycle continues to S phase • G2/M checkpoint monitors completion of DNA synthesis and DNA damage • If it fails, the cell cycle stops until the process(es) is/are corrected • M/G1 checkpoint monitors spindle formation and their attachments to kinetochores • If abnormal cycle, mitosis is arrested

5. The Cell cycle and its checkpoints Fig.1 Hoffman et al. 2005

6. p53 • The protein product it is involved in scrutiny of the cell cycle at G1/S checkpoint • If G1/S checkpoint detects DNA damage such as DNA double strand breaks (DSBs) and if the normal p53 gene is present the cell is programmed for apoptosis (cell death) and deleted from the cell population • If both copies of the gene p53 are mutated, abnormal functioning occurs, the cell may continue uncontrollable cell growing

7. ATM and Chk2 • ATM (ataxia telangiectasia mutated) is a protein is involved in detecting DNA damage (especially DSBs), interrupting with p53 the cell cycle when there is damage, and maintains normal telomere length • Chk2 is a kinase

8. BRCA1 • Breast cancer gene suppressor one • A gene located on chromosome 17 • It is necessary for cell cycle checkpoint, DNA repair, regulation of transcription, protein ubiquitination, and apoptosis to chromatin remodeling • Dysfunction causes cancer development

9. Link • Recent evidence suggest that two pathways can lead to BRCA1 phosphorylation in response to DSB breaks. DNA damage activates ATM and Chk2 kinases. These kinases phosphorylate BRCA1 and p53. The activated p53 protein arrests replication during S phase to allow DNA repair to take place. The activated BRCA1 protein participates in DNA repair with the BRCA2 protein, a protein called MRAD 51, and additional nuclear proteins

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

11. 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 (American type culture collection) cancer cell line

12. Data • Fig. 2. Determination of chromosomal HR in breast cancer cells.

13. Fig. 3. Determination of HR, radiation sensitivity, and S-phase checkpoint in BRCA1-transfected cells.

14. Fig. 4. Formation of Rad51 subnuclear foci in cells with or without exposure to IR.

15. Fig. 5. Determination of random chromosomal integration-linked NHR in NBS LBi fibroblasts and cells.

16. Fig. 6. Formation of Mre11 subnuclear foci in cells with or without exposure to IR.

17. Fig. 7. Influence of Chk2 kinase activity on NHR and HR.

18. Fig.8. Dual-role model of BRCA1 function in regulation of recombinational repair.

19. Results • We provide evidence that the promotion of HR and suppression of NHR are governed by the serine 988 residue of BRCA1, which is phosphorylated by the cell cycle checkpoint kinase Chk2. • Chromosomal HR is facilitated by BRCA1 (Fig. 3) • Chk2 protein kinase activity on BRCA1 is instrumental in dictating its functions (Fig. 3B and 7B and C). • The results presented in this paper raise the possibility that the promotion of HR mediated by the Chk2-BRCA1 pathway contributes to the prevention of breast cancer development.

20. HR may play a significant role in maintaining chromosome stability by occurring in a controlled manner relative to the activation of cell cycle checkpoints. • Surprisingly, ATM-mediated phosphorylation of serines 1423 and 1524 was not required for the function of BRCA1 in regulation of either HR or NHR in our study (Fig. 3 to 6). • An important finding in our study was that the serine 988 site of BRCA1 not only dictated the control of HR but also was critical for the inhibition of the MRN complex in vivo.

21. It is fascinating that BRCA1's role in regulation of both HR and MRN-dependent NHR is dependent on the Chk2 phosphorylation site. As HR and NHR events are likely to be very different processes, it suggests that BRCA1 exerts regulatory control of these processes at an early stage. Several lines of evidence support the notion that the relationship between BRCA1 and NHR is an active effect of BRCA1. • we propose a dual regulatory model of BRCA1 (Fig. 8), in which BRCA1, in conjunction with Chk2, promotes error-free HR while at the same time inhibiting putatively error-prone NHR associated with the MRN complex.

22. 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 phosporylation of BRCA1 regulates DNA double-strand break repair. Molecular and cell biology 24: 708-718. • Poznak C.V. and A.D. Seidman. 2002. Breast cancer. Encyclopedia of breast cancer, second edition. Elsevier science: 287-299.