Mechanism of homologous recombination driven by the intrinsically disordered domain of tumor suppressor PALB2 - Intrinsically disordered proteins (IDPs) lack a stable globular structure and are prevalent in the human proteome. They have many important biological functions, including serving as scaffold platforms that coordinate multiple proteins, forming membrane-less organelles, and acting as protein and nucleic acid chaperones. We discovered an unstructured protein domain within the PALB2 protein (Partner and Localizer of BRCA2) that recombines nucleic acid strands. Homologous recombination (HR) is a complex multistep reaction essential for all living organisms and directed by only a few known globular proteins. Recombination supported by the intrinsically disordered region (IDR) of PALB2 represents a novel function of IDPs and a new paradigm in nucleic acid metabolism. This proposal will establish the molecular mechanism of this novel IDP function. Sequence and structural properties of the PALB2 DNA-binding domain (DBD) and enrichment of eukaryotic DNA repair factors with disordered DBDs suggest a general hypothesis that similar regions evolved as a common tool to handle complex multistrand DNA and RNA intermediates during chromosome repair. We will investigate this hypothesis using the PALB2-binding domain of BRCA1. Results will be critical for establishing a functional role the PALB2-DBD IDR in DNA repair and will help to identify similar IDRs in other DNA and RNA metabolism factors. PALB2 is a central hub for large DNA repair complexes that mediates HR or a homology-directed repair (HDR) of chromosome breaks. PALB2 interacts with at least a dozen proteins, including breast cancer susceptibility proteins 1 and 2 (BRCA1, BRCA2) and the major homologous recombinase RAD51. We identified major DNA-binding amino acids in the PALB2-DBD that significantly contribute to DNA repair in cells. We discovered that PALB2-DBD is structurally disordered and can direct DNA or RNA strand exchange. We demonstrated that PALB2-DBD forms a compact dimer and condenses ssDNA. Thus, we hypothesize that a novel chaperone-like mechanism promotes PALB2-DBD-mediated strand exchange. Here, we will investigate this hypothesis using a set of complementary structural and spectroscopy methods (including single-molecule analysis) to determine conformational changes of PALB2-DBD and DNA. Furthermore, we will generate separation-of-function mutants for future studies of the physiological role of PALB2-mediated strand exchange in cells and will determine how the disordered PALB2-DBD regulates the activity of major recombinase RAD51. This work will establish the structural and mechanistic bases of PALB2 recombinational properties, will provide insights into the function of PALB2-mediated strand exchange in cellular DNA repair and genome maintenance, and will drive research of other scaffold proteins, such as BRCA1 and BRCA2, with intrinsically disordered DBDs. These results will yield unique insights into cancer etiology, aging, neurodegeneration, and other pathologies.
