In eukaryotic cells, DNA is packaged into chromatin. The basic structure of chromatin is the nucleosome, which is comprised of 146 base pairs of DNA wrapped around a protein octamer containing two molecules of each of the four core histones, H2A, H2B, H3, and H4. Both normal metabolic activities and environmental factors such as sunlight and industrial pollutants can cause damage to cellular DNA. To contend with the constantly occurring DNA damage, cells are equipped with multiple, highly conserved DNA repair pathways. One important DNA repair pathway, global genomic repair (GGR), is responsible for repairing bulky DNA lesions throughout the genome. It was discovered very recently that methylation of histone H3 lysine 79 (H3K79) by Dot1, a histone methyltransferase, is required for GGR. The goal of this project is to investigate how methylation of H3K79 by Dot1 mediates GGR by using the eukaryotic model organism brewer's yeast, Sacharomyces cerevisiae. Three questions will be addressed in this project. First, mono-ubiquitination of histone H2B lysine 123 and a basic patch in the N-terminal tail of histone H4 have been shown to trans-activate Dot1. The roles of these trans-activating elements in GGR and how they crosstalk with Dot1 in modulating GGR will be determined. Second, the nature of the GGR signal conferred by H3K79 methylation will be determined by changing the different states of H3K79 methylation in the cell. The roles of amino acids surrounding H3K79 on the nucleosome surface in GGR will also be determined. Third, GGR factors recruited by methylated H3K79 will be investigated by using a novel crosslinking technique that allows for detection of direct interaction between a rare protein and an abundant protein even if the interaction may be transient in living cells. New GGR-specific factors recruited by H3K79 will be identified by utilizing this novel crosslinking technique in combination with mass spectrometry.
Broader impacts: This project includes a significant educational component, with most of the funding used for research training of future professionals, including funding for graduate and undergraduate students to present their data at scientific meetings and publish results in scientific journals. Some of the new methods developed and findings generated from the project will be presented in the classroom. Unique yeast mutants and methodologies developed will be shared with the scientific community. Given the fact that Dot1, H3K79 methylation, nucleosome structure and the GGR process are highly conserved among eukaryotes, the findings generated from the yeast studies are likely to be applicable to other eukaryotic species, thus increasing the understanding of DNA repair processes, and the consequences of unrepaired damage.
