Mechanisms of DNA replication and maintenance in eukaryotes

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.

Grant Number: 5R35GM118129-10
NIH Institute/Center: NIH
Principal Investigator: PETER BURGERS