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.

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
The Cell Cycle

• 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

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

1. Hoffman I., K. Khanna, M. OConnel, N. Walworth,
   and T.J. Yen. 2005. Cell cycle checkpoints.
   Reactome org. lthttp//www.reactome.org/cgi-bin/eve
   ntbrowser?DBgk_ currentsID69620gt
2. Klug W.S., and M.R. Cummings. 2005. Essential of
   genetics, fifth edition. Pearson prentice hall
   26-27 and 497.
3. 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.fcg
   i?toolpubmedid14701743.
4. Poznak C.V. and A.D. Seidman. 2002. Breast
   cancer. Encyclopedia of breast cancer, second
   edition. Elsevier science 287-299.

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Questions?