Project 3: Structural and functional interplay of DNA Polymerase θ domains and other TMEJ factors

SUMMARY: PROJECT 3 This program project, “Polymerase theta, genome instability, and cancer”, will investigate mammalian DNA polymerase θ (Pol θ), the defining enzyme for repair of DNA double-strand breaks by theta-mediated end joining (TMEJ). Pol θ is a large protein (290 kDa in human cells) with a distinctive arrangement of an N-terminal helicase- like domain (HelD) and a C-terminal polymerase domain (PolD), connected by a long, mostly disordered central domain. Although work on Pol θ and TMEJ has made significant strides, the field still faces several limitations due to an incomplete state of knowledge. Using structural and biochemical methods, Project 3 (Structural and functional interplay of DNA Polymerase θ domains and other TMEJ factors) will determine how the different domains in Pol θ participate in DNA microhomology selection and repair. The unifying hypothesis is that these domains interact sequentially with DNA and other TMEJ protein factors in a coordinated fashion. Aim 1 will investigate the function of the central domain. Although this linker region is largely disordered, we uncovered that it contains several short regions that are well-conserved in chordates. Little is known about the function of these conserved regions. We will investigate how these regions are modified post-translationally because these modifications are relevant to breaks occurring during DNA replication. Aim 2 will investigate the structure and activity of the two folded HelD and PolD domains. We propose to investigate structure-informed and cancer-related variants of the human PolD and HelD domains bound to relevant DNA substrates using biochemistry and structural biology techniques. Aim 3 will determine biochemical mechanisms via reconstitution of TMEJ. We will define the minimal set of factors required to accomplish full reconstitution of TMEJ in vitro. The proposed work will be highly coordinated within the Program Project with the other three Projects and two Cores. Our combined diverse approaches, which include molecular biology, cancer cell biology, biochemistry, structural biology, and biophysics, work synergistically to answer questions related to the biological roles of Pol θ, the molecular mechanisms underpinning these roles, and how to harness this knowledge for improved cancer therapy. Proteins, DNA substrates, and experiments will be designed in consultation with Projects 1, 2, and 4 and monitored with feedback via Core A. Protein purification will be supported by Core B.