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Functional and Structural Interactions Between Rad51, Rad54, and Brca2 Proteins During Recombinational DNA Repair of DNA Double-Stranded Breaks
Functional and structural interactions between Rad51, Rad54, and Brca2 proteins during recombinational DNA repair of DNA double-stranded breaks.
Background: DNA double-strand breaks (DSBs) may result in chromosomal aberrations and can lead to cell death or uncontrolled cell growth (cancer). Homologous recombination (HR) is a DSB repair pathway and maintains the stability and integrity of the genome. The Rad52 epistasis group proteins form the core of the HR pathway, among which, Rad51, Rad54, and Brca2 proteins are critical components. Rad51 protein forms filaments on the ssDNA and performs homology search and DNA strand exchange. Rad54 stimulates the homologous pairing and heteroduplex DNA extension mediated by Rad51. Both proteins exhibit specific interactions that modulate their mutual functions. BRCA2 is one of two tumor suppressor genes that predisposes to breast cancer when mutated. Genetic, biochemical, and structural evidence supports a direct role of Brca2 protein in homologous recombination, but the exact function of the protein in this pathway remains unclear.
Objectives/Hypothesis: The objective is to understand the functional interactions between the human Rad51, Rad54, and Brca2 proteins through biochemical, molecular, and structural approaches. The hypothesis is that Rad54 and Brca2 compete for the same interaction surface on Rad51 protein, leading to a mutually exclusive interaction of both proteins with Rad51, that allows an ordered transition of molecular events at the Rad51 filament.
Specific Aims: (1) Generate and analyze human Rad54 interaction-deficient Rad51 mutants. (2) Generate and analyze compensatory Rad54 suppressor mutants that suppress interaction-deficient Rad51 mutants. (3) Analyze the effect of Brca2 and BRC peptides on the Rad51-Rad54 interaction. (4) Co-crystallize human Rad51-Rad54 (or Rad54 N-terminal interaction domain).
Study Design: (1) Generate interaction-deficient Rad51 mutants by screening (two hybrid system) and structure-informed approaches. (2) Purification and biochemical analysis of interaction-deficient Rad51 mutants in the presence and absence of Rad54 and/or Brca2/Brc peptides using in vitro assays for functional and physical interaction. (3) Generate compensatory Rad54 mutants by screening (two hybrid system) and structure-informed approaches. Biochemical analysis of the reconstituted Rad51:Rad54 interaction in functional assays in the presence and absence of Brca2/BRC peptides. (4) Crystallization of the Rad51-Rad54 co-complex in collaboration with an experienced crystallographer.
Potential Outcomes and Benefits of the Research: The expected results will provide molecular and possibly atomic details of the interactions between the human Rad51, Rad54, and Brca2 proteins. Sequential protein interactions have been proposed as critical for multi-step pathways. The proposed study will help to establish the far-reaching concept of shared protein interaction surfaces controlling consecutive, mutually exclusive interactions. Specifically, the results will further define the function of the Brca2 protein in homologous recombination, providing a molecular basis to understand the defects of some cancer relevant Brca2 mutations and possibly for therapeutic approaches.
Functional and structural interactions between Rad51, Rad54 and Brca2 proteins during recombinational DNA repair of DNA double-stranded breaks.
The human body is constantly exposed to DNA-damaging agents, such as ultraviolet light, ionizing radiation (IR) (from cosmos and terrestrial sources, diagnostic medical procedures, especially, during cancer radiation therapy), environmental chemicals, and its own toxic metabolic products. The damages can cause double-stranded breaks (DSBs) for DNA, the genetic material. Absence of or failure to accurately repair DNA DSBs can lead to cell death or uncontrolled cell growth - apoptosis or cancer. Homologous recombination (HR) is a specialized DNA repair pathway that provides error-free repair of DSBs.
A significant fraction of familial breast cancer cases is caused by mutations in the BRCA2 gene. A number of different models for the function of the Brca2 protein have been proposed. Recent genetic studies in mice and cell lines combined with studies of the Brca2 protein provided compelling evidence for a role in recombinational DNA repair of DSBs. DNA repair is a pivotal cellular process ensuring the integrity of the genome by processing DNA damage and restoring the original DNA sequence. DNA repair defects in humans are known to predispose to cancer, as shown in a variety of cancer susceptibility syndromes that affect a variety of different tissues, including the skin and the colon (xeroderma pigmentosum, HNPCC, ataxia telangiectasia, Bloom’s and Cockayne’s syndrome). The specificity of BRCA2 in causing breast (and ovarian) tumors may be a consequence of tissue-specific differences in the type and extent of the DNA damage that arises in these tissues.
The function of Brca2 protein is tightly intertwined with the central recombinational repair protein Rad51. Both proteins interact directly, and this interaction is started to be understood at atomic detail. However, the exact molecular function of Brca2 during Rad51-mediated recombinational DNA repair remains unclear. Rad51 protein is regulated by another crucial protein of HR, Rad54. The molecular functions of the Rad51 and Rad54 proteins are much better understood and can be reconstituted in cell free systems. Such systems are crucial for future drug development.
On the basis of structural information, we hypothesize that the Brca2 and Rad54 proteins compete for the same, shared protein interaction surface on Rad51. This suggests that the interaction of both with Rad51 might be mutually exclusive. Our objective is to understand the interaction between Rad51, Rad54, and Brca2 employing biochemical, molecular, and structural methods. The expected results will provide a more defined view on what the specific function of Brca2 is during recombination. Cancer relevant Brca2 mutations are found in its Rad51 interaction surface and our studies may provide insights into the molecular defect of such mutants. Moreover, protein interaction surfaces are large and provide potential drug targets to modulate the interaction, and hence function, of proteins. Our work aims at identifying such interfaces and demonstrating their importance for the functioning of recombinational DNA repair.