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Understanding the Tumor Suppression Function of BRCA1
Germline mutations of the tumor suppressor gene BRCA1 predispose women to breast and ovarian cancer. BRCA1 encodes a protein product of 1863 amino acids that contains an N-terminal Ring domain and C-terminal tandem BRCT motifs. BRCA1 participates in several fundamental nuclear processes including cell cycle checkpoint controls. It is believed that failure to activate DNA damage checkpoints leads to genomic instability and eventually tumorigenesis. Thus, intensive efforts have focused on dissecting the roles of BRCA1 in DNA damage checkpoints..
We have recently demonstrated that the BRCT domain at the C-terminus of BRCA1 is a phospho-protein binding motif. BRCA1 interacts with at least two cellular proteins, BACH1 and CtIP, through its C-terminal BRCT domains. Interestingly, the BRCA1/BACH1 complex and BRCA1/CtIP complex participate in different DNA damage checkpoint pathways.
Based on these initial observations, we hypothesize that different BRCA1 complexes carry out distinct functions in the cell and provide an explanation for the multi-functional nature of BRCA1. In this proposal, we will focus on the BRCA1/CtIP interaction and explore the regulation and the role of this complex in DNA damage responses.
We plan to:
I. Investigate the regulation of phosphorylation-dependent interaction between BRCA1 and CtIP.
II. Examine whether BRCA1/CtIP complex is required for the activation of downstream DNA damage checkpoint pathways.
III. Analyze missense BRCA1 BRCT domain mutants in BRCA1-dependent G2/M checkpoint control.
In Aim 1, we will study whether the BRCA1/CtIP interaction is cell cycle-regulated and/or regulated by DNA damage. In Aim 2, we will explore whether the interaction between BRCA1 and CtIP is required for CtIP phosphorylation following DNA damage. We will also assess the downstream DNA damage pathway that depends on BRCA1 and CtIP. In Aim 3, we will examine a collection of 38 missense mutations at the C-terminus of BRCA1 and assess whether these mutants affect the BRCA1/CtIP interaction and the checkpoint functions controlled by BRCA1.
Potential outcomes and Benefits of the Research:
The proposed study will not only provide mechanistic insight in the roles of BRCA1 in checkpoint controls, but will also uncover how mutations of BRCA1 disrupt its tumor suppression function.
Background: More than 50% of familial breast and ovarian cancer are due to mutations of BRCA1 or BRCA2 genes. The long-term goal of this research is to reveal the molecular mechanisms by which BRCA1 and BRCA2 operate as tumor suppressors. More than half of the mutations in BRCA1 lead to the deletions of BRCA1 C-terminal region (BRCT domain), suggesting that the C-terminus of BRCA1 is important for the tumor suppression function of BRCA1. We have shown recently that the C-terminus of BRCA1 interacts with phosphorylated proteins. We have evidence suggesting that using this property BRCA1 form multiple protein complexes and thus carry out distinct functions in responses to DNA damage. Therefore, the Objective/Hypothesis of this proposal is to understand mechanistically how BRCA1 participates in multiple DNA damage checkpoint pathways. Specific Aims: Specifically, we will examine how these BRCA1 complexes are regulated in specific aim 1. We will further dissect the DNA damage pathways controlled by these distinct BRCA1 complexes in specific aim 2. In specific aim 3, we will examine whether missense mutations at the C-terminus of BRCA1 affect BRCA1 function in checkpoint control. Study Design: We will study the cell cycle and/or DNA damage-dependent regulation of BRCA1 complexes in Aim 1. In Aim 2, we will assay for the activation of downstream DNA damage-signaling pathways in the presence or absence of BRCA1. In Aim 3, we will examine 38 missense mutations of BRCA1 and test whether some of them affect the function of BRCA1 in DNA damage checkpoint control. Many of these mutations are unclassified variants of BRCA1. There is no sufficient information regarding whether these mutations are associated with increased breast cancer risks. The functional assays we established here will help to determine whether certain 'unclassified' variants of BRCA1 confer high risks of breast cancer in women who carries them. Potential outcomes and Benefits of the Research: The study outlined here will not only improve our understanding of the tumor suppression function of BRCA1, but also will be of great help to the women who carry missense BRCA1 mutations in making sound clinical decisions based on solid scientific knowledge.