Research Grants Awarded
The Functions of Brca1 and Brca2 in Protecting Stalled DNA Replication Forks
Tumor Cell Biology V
Background: Brca1 and Brca2, two tumor suppressors frequently mutated in familial breast cancer patients, are both cruical for the maintenance of genomic stability. Although Brca1 and Brca2 clearly have distinct functions, they are both implicated in homology-directred repair (HDR), and in protecting DNA replication forks stressed/stalled by DNA damage or other interferences. The exact functions of Brca1 and Brca2 in protecing replication forks, however, remain elusive, mainly due to the lack of assay systems to locate and examine stalled replication forks. It was recently shown that inhibition of poly(ADP ribose) polymerase 1 (PARP1) selectively kills Brca1- and Brca2-deficient cells, suggesting a new strategy for breast cancer treatment. Nonetheless, the mechanism by which loss of PARP1 function eliminates Brca1/2 mutant cells remains to be determined. Objective: To understand the functions of Brca1 and Brca2 in protecting stalled DNA replication forks, we propose to use two novel strategies to stall and stress replication forks at specific sites in the genome. Using these new assay systems, we will directly examine whether Brca1 and Brca2 are important for the stability of replication forks at the stall sites. We will also address whether Brca1 and Brca2 control the repair of collapsed replication forks at these sites. Furthermore, using PARP1 as an example, we will investigate how the functions of Brca1 and Brca2 are affected by alterations of other proteins, and how Brca1- and Brca2-deficient cells can be specifically eliminated by interfering with DNA replication and DNA repair in these cells. Specific Aims: (1) Establish inducible assay systems to stall DNA replication forks in a site-specific manner. (2) Determine how loss of Brca1 and Brca2 affects the stability and the repair of stalled replication forks. (3) Examine whether Brca1 and Brca2 protect stalled replication forks from the instability induced by loss of PARP1 function. Study Design: Localized ADP-ribosylation of DNA and formation of DNA triplex will be used to stall replication forks in a site-specific manner. Pierisin1, a unique DNA ADP-ribosylase, will be fused with sequence-specific Zn fingers and conditionally expressed in cells. The target sequence of Zn fingers will be integrated to the genome to introduce site-specific replication blocks. In a parallel approach, triplex-forming single-stranded DNA will be generated in cells using a modified vector system, and targeted to specific sequences integrated in the genome. The engineered replication stall sites will be flanked by arrarys of binding sites of a fluorescent protein, so that the formation and repair of DNA breaks at these sites can be monitored. Furthermore, a direct repeats-green fluorescent protein (DR-GFP) reporter will be used at these sites to score DNA break formation and gene conversion. The stability and repair of replication forks stalled at these sites will be analyzed in cells deficient in Brca1, Brca2, and PARP1. Relevance: Understanding the critical functions of Brca1 and Brca2 in the maintenance of genomic stability will undoubtedly accelerate the eradication of breast cancer. The innovative approaches of this study will enable us for the first time to directly examine the functions of Brca1 and Brca2 in stabilization and repair of stressed/stalled replication forks. They will also allow us to determine how deficiency in these functions of Brca1 and Brca2 can be targeted by therapeutic strategies. The new methods proposed herein can be broadly applied to study how tumor suppressors protect the genome from replication stress.
Cancer, including breast cancer, are caused by mutations in genes that lead to uncontrollable cell proliferation. To avoid detrimental mutations in their genomes, cells must precisely duplicate their genomes everytime they divide, and efficiently repair DNA damage before they are converted to inheritable mutations. Duplication of the genome is accomplished by a large number of multi-protein machineries called replication forks. Since the faithful function of replication forks is constantly challenged by DNA damage and other impediments on chromosomes, cells have evolved mechanisms to stabilize stressed replication forks and repair broken replication forks once they appear. Many tumor suppressors, including prorteins involved in DNA repair and DNA damage signaling, have been implicated in this important process. Brca1 and Brca2 are two tumor suppressors frequently mutated in familial breast cancer patients. Although Brca1 and Brca2 appear to have distinic functions, they are both implicated in homology-directred repair, the main error-free DNA repair pathway that repairs DNA breaks, and in protecting DNA replication forks stressed/stalled by DNA damage or other interferences. Understanding these common functions of Brca1 and Brca2 will likely help to reveal how they protect people from getting breast cancer, and how breast cancer patients with mutations in these genes can be treated more effectively. To reveal the functions of Brca1 and Brca2 in protecting stressed DNA replication forks, it is essential to determine how loss of Brca1 or Brca2 affects replication forks that are under stress. However, it has been very difficult to examine the molecular events occurring at stressed replication forks because they usually do not take place at specific sites on DNA. In this study, we propose to use two novel strategies to stall replication forks at specific sites in the genome, so that the functions of Brca1 and Brca2 in protecting replication forks can be studied at these sites. Using the new assay systems, we will determine the exact roles of Brca1 and Brca2 in the stabilization and the repair of stalled replication forks. It is conceivable that in addition to Brca1 and Brca2, defects in other genes involved in protecting replication forks may also contribute to the susceptibility to breast cancer. When replication forks are rendered overly unstable, the cells bearing Brca1 or Brca2 mutations may be selectively killed. To assess these models, we will use the new assay systems to investigate how the functions of Brca1 and Brca2 are affected by alterations in other genes, and how Brca1- and Brca2-deficient cells can be specifically eliminated by manipulating functions of other genes, especially those involved in DNA replication and DNA repair. The results of this study will likely provide significant mechanistic insights to the functions of Brca1 and Brca2 in suppression of breast cancer. The innovative strategies of this study may be used to identify genes whose mutations elevate the susceptibility of breast cancer in people carrying Brca1 or Brca1 mutations, and to develop new therapeutic means to specific eliminate Brca1- or Brca2-deficieint breast cancer cells.