This proposal focuses on several DNA metabolic pathways in eukaryotes, with an emphasis on DNA replication and DNA damage response mechanisms. DNA polymerases ? (Pol ?) and ? (Pol ?) carry out the elongation and maturation of Okazaki fragments on the lagging strand of the replication fork, and translesion synthesis (TLS), respectively. Our recent discovery that their catalytic subunits contain an essential iron-sulfur cluster makes new functional studies of these enzymes both timely and important. Thus, we will determine whether the redox state of the iron-sulfur cluster affects the biochemical parameters of these enzymes, either in catalysis or complex interactions. We will test the hypothesis that regulated strand displacement synthesis by Pol ? is a critical aspect of its function during Okazaki fragment maturation. During this process, which occurs millions of times during each mammalian cell division, 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. Our proposed studies of mutagenesis focus on Rev1 and Pol ?, which through interactions with the replication clamp PCNA form a productive mutasome. The Rev1 protein serves as a scaffold onto which the TLS machinery is organized. We will test the hypothesis that posttranslational modifications act as on/off switches for mutagenesis by mediating functional interactions between Pol ? and Rev1 with PCNA. The primary focus of our checkpoint studies will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the DNA damage and replication checkpoints. The initiating step in these pathways is the activation of Mec1/ATR kinase activity through interaction with cell-cycle specific activators on single-stranded DNA substrates coated with the single-stranded binding protein RPA. Key features of these activators is that the activating regions are structurally disordered and contain small essential aromatic amino acid motifs. Biochemical studies are aimed at understanding Mec1 kinase activation by these proteins and complexes. In keeping with the MIRA principle, we also look forward to pursuing other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.

Public Health Relevance

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 damaged DNA and thereby introduce mutations. We also propose to study so-called cell cycle checkpoints, which monitor the intactness of our genetic material and the correctness of DNA duplication prior to cell division. 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 amenable to genetic and biochemical analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM118129-01
Application #
9071509
Study Section
Special Emphasis Panel (ZGM1-TRN-9 (MR))
Program Officer
Reddy, Michael K
Project Start
2016-06-01
Project End
2021-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
1
Fiscal Year
2016
Total Cost
$481,473
Indirect Cost
$165,753
Name
Washington University
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Stojkovič, Gorazd; Makarova, Alena V; Wanrooij, Paulina H et al. (2016) Oxidative DNA damage stalls the human mitochondrial replisome. Sci Rep 6:28942