Stress génotoxiques et Cancer

Research Overview: We are interested in DNA repair and its link to tumorigenesis. Using biochemistry, structural biology tools, and cell biology we focus on the study of BRCA2 (Breast Cancer Susceptibility Gene 2), a tumor suppressor protein involved in DNA repair.

DNA repair

The DNA inside the cell is continuously exposed to damage arising from exogenous sources such as ionizing radiation or endogenous sources such as byproducts of cell replication. All organisms have evolved different strategies to cope with these lesions. One of the most deleterious forms of DNA damage is called Double Stranded DNA breaks (DSB). In our cells, there are two major pathways to repair DSB: Homologous recombination (HR) and Non Homologous End Joining (NHEJ). HR is the most accurate mechanism to repair DSBs because it uses an intact copy of the DNA from the sister chromatid or the homologue chromosome to repair the break.

Homologous recombination

In humans, the central player of the HR process is RAD51; it catalyzes the DNA strand exchange that results in the repaired DNA. Through its interaction with RAD51, BRCA2 controls RAD51 function by locating it to the DSB. Thus, defects in BRCA2 lead to genomic instability, a hallmark of tumorigenesis.

Figure 1: Our current model for the role of BRCA2 in homologous recombination

Over my postdoctoral years, we have showed by biochemical and single molecule techniques how BRCA2 through a domain called the BRC repeats (represented in yellow) stimulates the formation of a stable nucleus of RAD51 on ssDNA by inhibiting RAD51 ATPase activity and at the same time blocking its non-productive assembly onto dsDNA allowing the correct orientation of the filament (Carreira et al., 2009), Figure 1. We have verified that this activity first described in a very small fragment of the protein (the BRC repeats) is actually representative of the whole BRCA2 protein (Jensen et al., 2010). By this mechanism, BRCA2 facilitates the subsequent steps of the HR process.

BRCA2 protein structure

Mutations in BRCA2 gene, cause predisposition to breast, ovarian and other types of cancer. As mentioned above, BRCA2 protein is required for the repair of DSB through homologous recombination.  BRCA2 has several functional domains to achieve its mediator activity (Figure 2).

Figure 2: BRCA2 functional domains: Schematic representation of BRCA2 protein showing its functional domains. H, helical domain; OB, oligonucleotide/oligosacharide-binding fold; NLS, nuclear localization signal.

The N-terminus is the most variable domain between species, the first 40 aa are conserved and have a transactivation domain, a PALB2 binding site (Partner and localizer of BRCA2) which is a protein that promotes BRCA2 localization in the chromatin, and an EMSY binding site, a protein that can negatively regulate the transactivation function of BRCA2. The BRC repeats occupy the central region of BRCA2, they consist of a cluster of 8 similar sequences of about 35 aa in length which are highly conserved between mammalian species and have the important ability to bind RAD51. The structure of BRC4 bound to RAD51 has been solved to atomic resolution (Pellegrini et al., 2002), (Figure 3).

Figure 3: Molecular surface and ribbon representation of RAD51 bound to BRC4 (PDB code 1n0w). RAD51 is shown in yellow and BRC4 in magenta. The highly conserved RAD51- interacting region of BRC4 is highlighted in red, including two residues that occupy the hydrophobic pockets of RAD51 (in stick representation).

Close to the central region, a DMC1 binding site has been described, which is the homolog of RAD51 in meiosis, implicating BRCA2 in meiotic recombination and, a FANCD2 binding site, a protein involved in the Fanconi Anemia pathway. The DNA Binding Domain (DBD) is also highly conserved and contains a a-helical motif, 3 domains with the oligonucleotide-binding (OB) fold and a tower structure emerging from OB2. These domains allow BRCA2 to bind ssDNA and dsDNA. DSS1 protein interacts with the DBD and it has been shown to be important for BRCA2 stability. The extreme C-terminus contains 2 nuclear localization signals (NLS), which serve to recruit RAD51 to the nucleus and an additional RAD51 binding motif that appears to regulate the interaction with RAD51 in a cell cycle-dependent manner.

In addition, BRCA2 can associate with other proteins and participate in related pathways such as cell cycle control and embryo development. As a consequence, a defect in this protein leads to uncontrolled cell replication, and genomic instability, both hallmarks of tumor formation.

Projects 

The main projects of the lab include:

1) Reveal new roles of BRCA2 by mapping the interactions with other proteins: BRCA2 comprises 3,418 aa. This protein is known to interact with several partners such as RAD51 or PALB2 however; there are regions of the protein still unexplored. The study of these partner proteins is very interesting because it can reveal new bridges between different DNA repair or related pathways for example, recently, a DMC1 binding site was found in BRCA2 implicating BRCA2 in meiotic recombination (Thorslund et al., 2007).

Similarly, the study of the evolutionary conservation of domains in BRCA2 can give us clues on the  functional relevance of certain regions of the protein still unknown.  For instance, the BRC repeats content of different organisms varies from 1 to 15 however, all the BRCA2-like proteins seem to function in a similar manner.

2) Exploit regions of the protein as a therapeutic tool for tumor treatment.

Mutations in BRCA2 cause deficiency in the DNA repair by HR. The inhibition of the alternative DNA repair pathway that takes over the repair process in this situation results in selectively killing tumor cells. Some of these so called synthetic-lethal compounds are currently under clinical trial in anticancer therapy (ex. PARP inhibitors).

Figure 4: Scheme showing the action of PARP inhibition and the consequences of becoming resistant to this inhibition: SSB, Single Strand DNA Breaks; DSB, Double strand DNA breaks; HR, Homologous Recombination.

Because they are genetically unstable, these tumors eventually acquire secondary mutations that make them resistant to the treatment (Figure 4). We will develop new strategies to block interactions between key proteins in the HR pathway in order to sensitize tumors cells to radio or chemotherapy.

3) Explore different ways to target inhibitor molecules to the tumor cells.

One fundamental problem that anticancer therapies with peptides or small drugs face is their poor performance pharmacologically. Limitations include low stability in the plasma, bioavailability and poor tumor cell penetration. In our lab, we plan to develop new strategies to cope with these problems.