- Bergisch Gladbach
- [email protected]
Mechanism of Genome Protection by Homologous Recombination Repair: A Single Molecule DNA-Protein Interaction Study
Biotechnology with specialization in medical research
First level degree in Job Creation Oriented Biotechnology
German high school degree
Radiation therapy and several chemotherapeutics exploit the induction of double-stranded DNA breaks (DSBs) to trigger cell death. Tumor resistance to treatment can arise from an increased or re-acquired capacity to repair DSBs by homologous recombination (HR). Elevated recombination proficiency correlates with high levels of the recombinase protein (RAD51) in tumors. For successful recombination RAD51 needs specific interactions with BRCA2, a tumor suppressor gene frequently mutated in hereditary breast and ovarian cancer. Non-functional BRCA2 results in recombination deficient phenotypes, which are exploited in treatment with PARP inhibitors that induce synthetic lethality in HR deficient cells. Some tumors develop resistance to PARP inhibition by reverting mutations in BRCA2 and thus restoring DSB repair by HR. We propose that peptide or small molecule inhibitors specifically targeting the interactions between RAD51 and BRCA2 will greatly sensitize tumors to DSB inducting therapies and, importantly, re-sensitize tumors to therapy that acquired resistance due to restoration of HR. Our peptide inhibitor design is based on combinatorial mutagenesis on a BRCA2 fragment that according to the structure of RAD51-BRC4, would bind the same interface region of RAD51 as BRCA2, but with increased affinity. Candidate peptides will be selected by phage display. Another approach is the screening for small molecules that inhibit RAD51-BRC binding (in silico screen based on structure) or disrupt HR. To find compounds disrupting HR we aim to employ a synthetic lethal screen using PARP inhibitors. We aim to deliver successful candidate peptides specifically to tumors either by exploiting the natural oncotropism of autonomous parvovirus or in combination with tumor-penetrating iRGD peptides. Small molecule delivery will be assessed in combination with iRGD peptides. This way, HR should remain intact in healthy cells while tumors are specifically sensitized to treatment.
The overall goal of our research is to understand the mechanisms and biological function of complex genome transactions such as homologous recombination. Homologous recombination, the exchange of sequences between homologous DNA molecules, is essential for accurate genome duplication, DNA damage repair and chromosome segregation. Recent advances in fluorescence detection and microscopy as well as our success in producing fluorescently labeled functional recombination proteins make it possible to analyze the mechanistic details of homologous recombination at the single molecule or single complex level. Single molecule analysis provides information on intermediate states, functional and structural variability and the distribution of variable states that cannot be recovered from bulk biochemical assays. Biologically, the importance of a reaction, such as homologous recombination, lies in the result of one such event occurring in the complex molecular environment of a cell. Thus the random variation in the details of molecular behavior, that we can now determine with single molecule mechanistic studies are of great importance for understanding how relatively simple biochemical activities are combined to create complex and adaptable living systems.