DESCRIPTION: This proposal is centered on studies of the mechanism of DNA replication in eukaryotic cells (1) and on the consequences of replication dysfunction with regard to spontaneous and damage- induced mutagenesis (2), and to cell cycle checkpoint activation (3). Our primary approaches combine biochemical and biophysical analysis with genetic analysis in the yeast Saccharomyces cerevisiae, to gain insight in each of these three broadly defined pathways and their interconnectivity. Our studies are augmented with structural studies of the complex machineries that function in these processes. Proposed DNA replication studies are based on a strong record of progress in defining mechanisms of lagging strand DNA replication and Okazaki fragment maturation. Since Okazaki fragments represent by far the most frequent DNA discontinuities in all cells, it is imperative to understand the different layers of regulation of this process. Okazaki fragment maturation has primarily been studied in well-defined biochemical systems, in isolation from other events that occur at the replication fork. We propose to expand our studies within the context of a complete replication fork, which has been assembled at a yeast replication origin. Our mutagenesis studies will center on the main actors, DNA polymerase z and Rev1. On the one hand, Rev1 promotes DNA lesion bypass by Pol z; on the other hand, it limits the extent of mutagenesis by inhibiting Pol z-dependent DNA synthesis outside the narrow environment of the lesion. We will unravel the mechanism that underlies this dual regulatory function of Rev1. The primary focus of our checkpoint studies is on the two sensor protein kinases Mec1 and Tel1, the orthologs of human ATR and ATM, respectively. Biochemical and genetic studies will be combined with cryo-EM studies to understand how the basal activities of these unique protein kinases are activated. Furthermore, the advantage of having an efficient DNA replication system available will allow us to begin addressing the coupling between replication arrest and the downstream response pathways. Finally, in keeping with the MIRA principle, we will pursue other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.