1. COMPUTING OF THE RAD51 -BRCA2 INTERACTIONS Assist. Prof. Albena Todorova, PhD Andrey Kirov, Msc Peycho Petkov, PhD Computing of the effects of genetic mutations in the BRCA2 gene to the protein structure and evaluation of these changes to their interactions with other proteins

2. WHY? 519 000 women died in 2004 due to breast cancer, and although breast cancer is thought to be a disease of the developed world, a majority (69%) of all breast cancer deaths occurs in developing countries (WHO Global Burden of Disease, 2004)‏ According to WHO breast cancer is the most common cancer in women worldwide, comprising 16% of all female cancers Breast cancer survival rates vary greatly worldwide, ranging from 80% or over in North America, Sweden and Japan to around 60% in middle-income countries and below 40% in low-income countries (Coleman et al., 2008). The low survival rates in less developed countries can be explained mainly by the lack of early detection and prophylactics programmes

3. Family history of breast or ovarian cancer - increases the risk by a factor of two or three. Some mutations, particularly in BRCA1 and BRCA2 genes result in a very high risk for breast cancer RISK FACTORS Age – cancer risk increases with aging, no matter of genetic predispositions Hormone influence – high estrogen level increases the risk Obesity Nutrition diete

4. DNA ISOLATION BRCA GENETIC TESTS ALGORITHM SEQUENCING OF BRCA1 & BRCA2 GENES qPCR/MLPA ANALYSIS OF BRCA1 & BRCA2 GENES NORMAL RESULT EVERYTHING IS OK LARGE DELETIONS/DUPLICATIONS ARE DETECTED INCRESED RISK OF BREAST AND OVARIAN CANCER MUTATION WITH KNOWN GENOTYPE- PHENOTYPE CORRELATION DETECTION OF POINT MUTATIONS MUTATION WITH UNKNOWN GENOTYPE- PHENOTYPE CORRELATION ?

5. BRCA2 The BRCA2 tumour suppressor is essential for homologous DNA recombination, a process for the errorfree repair of DNA double-stranded breaks, in the cells of higher eukaryotes BRCA2 central function during recombination is to control the RAD51 recombinase, which is indispensable for DNA recombination The homologous DNA recombination begins when broken DNA is end- resected to generate ssDNA, upon which RAD51 oligomerizes to form an active nucleoprotein filament. The RAD51 nucleoprotein filament catalyzes strand exchange by invading and pairing with a homologous DNA duplex, used as a template for repair

6. Reymera et al., 2009

7. BRCA2-RAD51 INTERACTIONS Two distinct regions in human BRCA2 bind directly to RAD51: 1.Eight BRC repeats, evolutionarily conserved motifs of approximately 35 residues each, distributed in a 1100 residue region of BRCA2 (BRC1-8) - conserved amongst vertebrate orthologues in both their sequence and spacing 2. C-terminus RAD51-interacting region - stabilizes RAD51 oligomeric assemblies in vitro both on and off DNA

8. BRCA2-RAD51 INTERACTIONS BRCA2 BRC repeats

9. SIMULATION OF BRCA2-RAD51 INTERACTIONS PROBLEMS The whole 3D structure of BRCA2 is still unknown, we have information for the 3D structure of region of BRC4 repeat: Aminoacid sequence of BRC4 repeat: PTLLGFHTASGKKVKIAKESLDKVKNLFDEKEQ Position: 1524 1546

10. Crystal structure of a RAD51-BRCA2 BRC4 repeat complex DOI:10.2210/pdb1n0w/pdb Source: RCSB Protein Data Bank

11. SIMULATION OF BRCA2-RAD51 INTERACTIONS PROBLEMS In this region we have very limited information about the correlation Genotype - Phenotype Solution In this region we have 1 mutation with known correlation Genotype- Phenotype and used it to test our model Computing the interaction of the normal/wild type BRC4 and RAD51 and its use as a base to compare

12. Mutation № MutationSR-LJCoul-SRSR-Energy 1 Wild type-322.345-376.139-698.484 2 Trp1520Ala-381.386-409.69-791.076 3 Phe1524Val-325.782-319.335-645.117 4 Gly1529Arg-365.424-341.879-707.303 5 Pro1519His-306.214-307.031-613.245 6 Pro1519Lys-377.373-411.534-788.907 7 Leu1521Ser-301.457-264.946-566.403 8 Phe1524Tyr-336.86-359.267-696.127 9 Phe1524Trp-256.996-339.593-596.589 10 Phe1524Gly-260.885-220.265-481.15 11 Phe1546Tyr-469.893-494.114-964.007 12 Phe1546Trp-305.869-294.978-600.847 13 Phe1546Ser-331.077-355.703-686.78 14 Asp1547Tyr-360.436-348.169-708.605 SIMULATION OF BRCA2-RAD51 INTERACTIONS Normal Phenotype

14. CONCLUSION THE STRUCTURAL HOMOLOGY IN THE REGION OF THE TWO HIDROPHOBIC POCKETS IS MORE IMPORTANT FOR THE BRC4- RAD51 COMPLEX STABILITY THAN THE HIDROPHOBIC INTERACTIONS INSIDE THE POCKET

15. CONTROVERSARY Position: 1524 1546 A peptide containing both F1524 and F1546 mutated to tryptophan retains similar behaviour

16. SO? WHAT TO DO?

17. MORE RESEARCH

18. Acknowledgement: Financial support of Bulgarian Supercomputing Centre (BGSC) is greatly appreciated – Grant 10/2009