**3.1 The BRCA1 structure – function relation paradigm**

The Breast Cancer Susceptibility Gene 1 protein (BRCA1) is a multifunctional nuclear phosphoprotein of 1863 residues (220-240 kDa). BRCA1 was attributed the role of a tumor suppressor involved in multiple cellular functions (Starita & Parvin, 2003). Most of BRCA1 is located in the cell nucleus and is phosphorylated in a cell cycle-dependent manner by a number of kinases (reviewed by Ouchi, 2006). Depending on the position and the number of phosphorylated residues, BRCA1 participates in different multiprotein complexes performing diverse tasks. Therefore, BRCA1 has been implicated in a variety of functions required for the maintenance of genomic stability (Rowling et al., 2010).

Regarding DDR, BRCA1 has been attributed many roles in regulation of genome integrity including DNA replication, cell cycle checkpoint control, apoptosis, regulation of transcription, chromatin unfolding and protein ubiquitination. The ascribed functions are exerted through an extensive number of protein interactions reported (Jasin M. 2002 as cited in Murphy & Moynahan 2010). In brief, upon detection of chromatid relaxation due to breakage of both strands of the double helix of DNA, BRCA1 – being activated by ATR kinase – is recruited to the damage breakpoint assisting assembly of the BRCA2 – RAD51 complex in order to replace RPA and restore damage by the high fidelity process of HRR. In parallel, BRCA1 interaction with Fanconi Anemia (FA) and other complexes regulates G1/S and G2/M checkpoints. BRCA1 implication in cell cycle regulation is assisted by complex formation with BRCA1 interacting protein C-terminal helicase (BRIP1) and CtIP which are activated in S-phase by post-translational modifications. A graphical representation of the BRCA1 protein, including sites of both post-translational modifications and regions involved in protein-protein interactions, is depicted in Fig 1. The amino-terminal region of BRCA1 contains a distinct ~100aa RING finger motif involved in ubiquitin ligase activity and enables BRCA1 to mono- or poly-ubiquitinate cellular proteins. BARD1 (another RING and BRCT domain-containing protein) is the 'permanent' partner of BRCA1 in the formation of the ubiquitine ligase complex. Phosphorylation of specific residues of BRCA1 appears to regulate its participation in transcription regulation and ubiquitination of substrate proteins. As many different BRCA1 species are produced by alternative splicing of its mRNA, the phosphorylated residues each form contains may

Structure-Function Relationship of DNA Repair

al., 2001; Mark, 2005; Ouchi, 2006).

**3.2 BRCA1 structure modifications found in cancer** 

probability rate of 45-60% and 20-40%, respectively.

(Glover, 2006).

ovarian cancers.

Proteins: Lessons from BRCA1 and RAD51 Studies 111

Analysis of the BRCA1 mutational database (BIC, http://research.nhgri.nih.gov/bic/) indicates that both RING and BRCT repeats are most frequently mutated in women at risk of cancer, and have been further studied. Many research groups have used structural and biochemical methods to probe the function of BRCA1 and characterize the plethora of unclassified variants identified in breast cancer patients found in BIC database. Among the hundreds of distinct mutations uncovered in BRCA1, for the vast majority, there is insufficient genetic linkage data to determine the cancer risk associated with them

The central region of BRCA1, between the two terminal domains, bears relatively low sequence identity between mammalian BRCA1 homologs, and attempts to define structured domains within this region indicate that this part of BRCA1 is largely unstructured (Glover, 2006; Mark, 2005). This region is extensively phosphorylated by DNA damage-associated kinases like ATM and may serve as a phosphorylation-dependent docking site for other proteins involved in the DNA damage response, or even for damaged DNA itself (Paul et

Mutations in brca1 and brca2 genes have been found in 30-50% of hereditary breast and ovarian cancers. Women carrying BRCA1 mutations are particularly susceptible to the development of breast or ovarian cancer at an age earlier than 35-40 years old with a

Most cancer-associated BRCA1 mutations identified so far, result in the premature translational termination of the protein and influence BRCA1 integrity and function. A large number of missense mutations is located in BRCT tandem repeats of BRCA1, while only few of them may cause loss of the protein's function, abolition of protein interactions and protein miss-localization. Therefore, it seems that the BRCT repeats in BRCA1 are essential for the tumor suppressing function of the protein as protein truncation and missense variants within the BRCT domain have been shown to be associated with human breast and

Variants that result in large truncations are deleterious to function and therefore can be classified as disease-associated. In contrast, missense mutations typically remain unclassified. Thus, the BIC database currently contains more than 108 missense mutations in the BRCT domains of BRCA1, but only 7% of them have been classified. These missense mutations may be either polymorphisms or mutations predisposing the carrier to cancer progression. The variants D1692Y, C1697R, R1699W, A1708E, S1715R, P1749R and M1775R all appear to be associated with an increased risk of breast cancer, while M1625I appears to be a benign polymorphism (Williams et al., 2003). Unfortunately, most of the missense mutations could not been assessed for disease association. An attempt to classify these variants by measuring the thermodynamic stability of the BRCA1 BRCT domains resulted in investigation of the effects of 36 missense mutations (Rowling et al., 2010). The mutations show a range of effects. Some do not change the stability, whereas others destabilize the protein by as much as 6 kcal mol−1; one-third of the mutants were considered to destabilize the protein by an even greater amount, as they could not be expressed in soluble form in *Escherichia coli*. Several computer algorithms were used in an attempt to predict the mutant effects. According to these results the variants were grouped into two classes (destabilizing by less than or more than 2.2 kcal mol−1). Importantly, with the exception of the few mutants located in the binding site, none showed a significant reduction in affinity for phosphorylated substrate. These results indicate

regulate different functions. Moreover, the balance between full length and spliced forms of BRCA1 may play an important role in tumor suppression (Ouchi, 2006).

Fig. 1. Primary structure of BRCA1 Tumor Suppressor Protein. The phosphorylation sites and and its binding partners are indicated.

The carboxy-terminal domain of BRCA1 contains two structurally identical BRCT (**BR**CA1 **C**-**t**erminal) tandem repeats each containing ~90 amino acid residues. BRCT domains are found in proteins involved in DNA repair and maintenance of genomic stability, and more recently, the BRCT repeat has been recognized as a phosphopeptide-binding domain. The structure of each repeat consists of a parallel four-stranded β-sheet located at the central part of the domain surrounded by three α-helices (Fig. 2). The two BRCT repeats fold together in a specific head-to-tail manner, giving rise to the formation of a conserved, almost allhydrophobic, inter-repeat interface, forming a phosphopeptide binding pocket. BRCT like domains have also been found in BRCA1 interacting proteins such as 53BP1 and BARD1. BRCT repeats are a family of phosphopeptide binding domains implicated in DNA damage response. Therefore, BRCTs are considered as protein-docking modules involved in eukaryotic DNA repair. Although BRCTs are characterized by low sequence homology they retain a generally well-conserved structure organization.

Fig. 2. Ribbon representation of the BRCA1-BRCTstructure. Positions of selected cancer– related mutations are indicated. M1775K and M1783T are located at the inter-BRCT-repeat interface where the BRCA1-BRCT binding groove for Phe 13 is also located. The exposed V1696L is located at the N-terminal BRCTstructural repeat. V1809F, P1812A are found at the C-terminal BRCT repeat. The positions of missense mutations from previously published studies are also depicted. (From Drikos et al., 2009).

regulate different functions. Moreover, the balance between full length and spliced forms of

Fig. 1. Primary structure of BRCA1 Tumor Suppressor Protein. The phosphorylation sites

Fig. 2. Ribbon representation of the BRCA1-BRCTstructure. Positions of selected cancer– related mutations are indicated. M1775K and M1783T are located at the inter-BRCT-repeat interface where the BRCA1-BRCT binding groove for Phe 13 is also located. The exposed V1696L is located at the N-terminal BRCTstructural repeat. V1809F, P1812A are found at the C-terminal BRCT repeat. The positions of missense mutations from previously published

The carboxy-terminal domain of BRCA1 contains two structurally identical BRCT (**BR**CA1 **C**-**t**erminal) tandem repeats each containing ~90 amino acid residues. BRCT domains are found in proteins involved in DNA repair and maintenance of genomic stability, and more recently, the BRCT repeat has been recognized as a phosphopeptide-binding domain. The structure of each repeat consists of a parallel four-stranded β-sheet located at the central part of the domain surrounded by three α-helices (Fig. 2). The two BRCT repeats fold together in a specific head-to-tail manner, giving rise to the formation of a conserved, almost allhydrophobic, inter-repeat interface, forming a phosphopeptide binding pocket. BRCT like domains have also been found in BRCA1 interacting proteins such as 53BP1 and BARD1. BRCT repeats are a family of phosphopeptide binding domains implicated in DNA damage response. Therefore, BRCTs are considered as protein-docking modules involved in eukaryotic DNA repair. Although BRCTs are characterized by low sequence homology they

BRCA1 may play an important role in tumor suppression (Ouchi, 2006).

and and its binding partners are indicated.

retain a generally well-conserved structure organization.

studies are also depicted. (From Drikos et al., 2009).

Analysis of the BRCA1 mutational database (BIC, http://research.nhgri.nih.gov/bic/) indicates that both RING and BRCT repeats are most frequently mutated in women at risk of cancer, and have been further studied. Many research groups have used structural and biochemical methods to probe the function of BRCA1 and characterize the plethora of unclassified variants identified in breast cancer patients found in BIC database. Among the hundreds of distinct mutations uncovered in BRCA1, for the vast majority, there is insufficient genetic linkage data to determine the cancer risk associated with them (Glover, 2006).

The central region of BRCA1, between the two terminal domains, bears relatively low sequence identity between mammalian BRCA1 homologs, and attempts to define structured domains within this region indicate that this part of BRCA1 is largely unstructured (Glover, 2006; Mark, 2005). This region is extensively phosphorylated by DNA damage-associated kinases like ATM and may serve as a phosphorylation-dependent docking site for other proteins involved in the DNA damage response, or even for damaged DNA itself (Paul et al., 2001; Mark, 2005; Ouchi, 2006).
