Employing polymerase usage sequencing to better understand fundamental aspects of eukaryotic replication

DNA replication represents an essential mechanism which ensures short-term survival of all organisms as well as propagation of life in general. In this thesis, we explore fundamental aspects of eukaryotic replication and develop techniques which provide better performance and allow comprehensive analysis of understudied phenomena. In chapter 1, we summarise our attempts to improve standard polymerase usage sequencing protocol. In chapter 2, we test the hypothesis that increased Polδ levels interfere with canonical leading strand replication. In chapter 3, we explore replication dynamics in mutants depleted of senataxin RNA/DNA helicases Sen1+ and Dbl8+. In chapter 4, we present a system to study replication of induced heterochromatinised domains. In chapter 5, we characterise basic properties of small Polδ subunit Cdm1+.

Thesis organisation

The Presented work composes of 8 distinct sections including introduction, methods, chapters 1-5 and conclusions. The introduction section introduces general concepts relevant to the main theme of the thesis. The methods section provides comprehensive description of procedures, reagents and biological material used in presented experiments. Chapters 1-5 cover particular research projects developed in the lab. Each chapter provides a specific theoretical background together with description and discussion of acquired results. The conclusion section summarises presented work.