DESCRIPTION: The four eukaryotic B-family DNA polymerases (Pol ?, ?, ?, and ?) are multi-subunit enzymes that carry out DNA replication and translesion synthesis (TLS). Our recent discovery that their catalytic subunits contain an essential iron-sulfur cluster makes new functional studies of these enzymes both timely and important. This proposal focuses on Pol ? and Pol ?. Pol ? carries out the synthesis and maturation of Okazaki fragments on the lagging strand of the replication fork, and is also responsible for DNA synthesis in recombination and repair processes. Pol ? is essential for translesion synthesis (TLS) and mutagenesis when DNA replication forks stall due to DNA damage or replisome dysfunction. The proposed studies of these two DNA polymerases are central to testing several hypotheses that address unsolved problems in DNA metabolism. The first is that posttranslational modifications act as an on/off switch for mutagenesis by mediating functional interactions between Pol ? and Rev1 (aim 1). Rev1 protein serves as a scaffold onto which the TLS machinery is organized. Preliminary studies indicate that phosphorylation of Rev1 dramatically activates Pol ?-mediated TLS. An integrated biochemical and genetic approach will be used to determine how phosphorylation of Rev1 activates TLS through modulating interactions with Pol ? and with other factors such as the replication clamp PCNA. The second hypothesis is that closely regulated strand displacement synthesis by Pol ? is a critical aspect of its function during Okazaki fragment maturation (aim 2). During this process, which occurs millions of times during each mammalian cell division, precisely regulated strand displacement synthesis by Pol ? generates 5'-flaps that are cut by the flap endonuclease FEN1. The kinetic mechanism of this machinery will be determined, and their physiological relevance will be queried through genetic analysis of informative mutants. Finally, based on preliminary data showing that the iron-sulfur cluster of Pol ? undergoes redox chemistry under physiologically relevant conditions, we hypothesize that a change in the redox state of the cell, due to oxidative stress, results in a change of the redox state of the enzyme, and of its activity (aim 3). Using solution chemistry and electrochemistry, the iron-sulfur cluster of Pol ? will be converted into different redox states, and the consequences for its enzymatic activities, and interactions with subunits and accessory factors will be studied.
The correct replication of cellular DNA is crucial in order to maintain the integrity of our geneti information. In this application, we propose to study the DNA polymerases that replicate our DNA with high fidelity, and those that replicate DNA in response to DNA damage and that can lead to the generation of mutations and to cell death. Patients with known defects in the proper response to DNA damage are at an increased risk for developing cancer. These pathways are highly conserved, from human to yeast. We propose to use yeast as an experimental model organism, because this organism is more approachable to genetic and biochemical analysis.
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