Assembly and activation of DNA damage sensing kinase Mec1-Ddc2

This thesis comprises five chapters.
Chapter 1 presents the introduction. It summarizes the role of checkpoint kinase Mec1-Ddc2 in the DNA
damage response. In chapter 1, an extensive account of the literature on Mec1-Ddc2 recruitment and
activation at DNA damage sites is provided which sets the foundation for chapter 2.
Chapter 2 presents results that contribute the major finding of my thesis. This chapter focuses on how
homodimers of Ddc2 are recruited to DNA damage sites via interaction with RPA. Based on structural,
biochemical and in vivo data, the chapter presents a to-scale model of Mec1-Ddc2 bound to ssDNA-RPA
complexes at DNA damage sites and shows that cell survival after UV-damage is dependent on Ddc2
homodimerization and recruitment to RPA. These results are published in Deshpande et al., Molecular
Cell, 2017.
Chapter 3 presents experimental results which show that an N-terminal region within the coiled-coil
domain of human ATRIP is important for coiled-coil homodimerization.
Chapter 4 presents results on the role of the MRX protein complex as a structural linchpin that holds
sister chromatids together at DNA double-strand breaks. This function of MRX is apparently dependent
on its interaction with a domain in RPA that also binds Ddc2. The results are published in Seeber et al.,
Molecular Cell, 2016.
Chapter 5 summarizes the major conclusions of this thesis and discusses the future directions.