Human DNA Replication Machines: Structure-function Studies

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Project Summary/Abstract The genome replication is fundamental process of life that impacts virtually every aspect of human health. Therefore, a detailed understanding of genome replication mechanisms is vital for future advances in disease diagnosis, drug design, and patient treatment. The bulk of human DNA replication is performed by the B-family DNA polymerases Polδ and Polε. However, Polδ and Polε cannot begin synthesis without DNA primers. To circumvent this problem, a specialized RNA polymerase called primase generates the initial primers. Then, a dedicated B-family DNA polymerase α (Polα) working in a tight complex with primase (referred to as a primosome) extends the RNA primers with deoxyribonucleotides, before switching them to Polε for the start of leading-strand replication and to Polδ for the start of replication of each of the millions of Okazaki fragments of the lagging strand. The remaining member of the B-family is DNA polymerase ζ (Polζ), which is a key player in translesion DNA synthesis. A significant gap remains in the current understanding of B-family DNA polymerases' function, especially regarding the key factors that tightly coordinate polymerase transactions at the replication fork. The crucial components of this global coordination are the mechanisms of template:primer handover from Polα to Polε and Polδ during asymmetrical synthesis of both the leading and lagging strands, counting the length of Okazaki fragments, and the switch of Polδ and Polε to productive elongation. We discovered that the accessory B-subunit of Polδ also makes a complex with the catalytic subunit of Polζ, which is important for the polymerase switch during lesion bypass. However, the mechanism of this switch remains unknown. One of the biggest impediments in resolving these challenges is insufficient structural information, especially for entire polymerase complexes, including Polδ, Polε, and Polζ, as adequate knowledge of molecular structure is essential for the design of meaningful functional assays. Based on our previous productive studies of primosome and the components of Polδ, Polε, and Polζ, we propose a new direction of investigation that examines the tightly coordinated events in primer synthesis, primer handoff from Polα to Polε and Polδ, and their switch to accurate elongation mode. For the proposed studies, we will apply X-ray crystallography and a variety of structure-guided biochemical and single-molecule experiments. Most of these studies will be conducted using the in vitro reconstituted human replisome.