Structural and biochemical studies on DNA packaging and replication

Abstract

In the eukaryotic nuclei, DNA is packaged into chromatin fibers containing repeating units of nucleosomes. Each nucleosome is composed of an octamer of histone proteins wrapped by DNA. Nucleosomes along the fiber interact in various ways leading to the formation of higher order structures. These higher orders structures play an important role in the regulation of DNA accessibility. The goal of this part was to gain insight into how nucleosomes interact in chromatin fibers. This study resulted in a new crystal structure of a 157 bp nucleosome repeat length tetranucleosome at 6.7 A resolution. Comparison of the interaction between nucleosomes observed in this structure and previously solved oligonucleosome structures revealed a diversity in the modes of how nucleosomes interact in chromatin fibres. I have developed biochemical tools that serve to detect the diversity of these interactions and to capture the structural heterogeneity in chromatin fibres. A second part of the thesis resulted in the structure of the DNA binding domain of the fission yeast protein Sap1 which was solved to 1.4 A resolution . The structure gives insights into Sap1 function in DNA replication.