Multiple laser lines that are used for single-molecule TIRF (total-internal fluorescence) imaging

Hasan Yardimci : Architecture and dynamics of the eukaryotic replication machinery

Introduction

An essential component of the replisome complex is the polymerase, which synthesises new DNA on unwound strands.

First, a primase complex associates with unwound DNA and synthesizes DNA/RNA primers. Although the DNA primer at the leading strand is extended continuously, the lagging strand is synthesised discontinuously as Okazaki fragments. It is important to understand how different poymerases coordinate DNA synthesis for accurate replication. Live cell imaging in bacteria showed that a single replisome contains three polymerases, one acting on the leading strand and two on the lagging as opposed to the previous assumption that there is one polymerase acting on each strand. The presence of two lagging strand polymerases was shown to be important for processive lagging strand synthesis.

Unlike prokaryotes, eukaryotes employ different polymerases to synthesise leading and lagging strands. Upon priming by polymerase alpha-primase complex (pol α), the leading strand is replicated by polymerase epsilon (pol ε) while the lagging strand is replicated by the action of polymerase delta (pol δ) (Figure 1). Currently, little is known about the stoichiometry and dynamics of eukaryotic replisome components including polymerases. How long does pol α remain on DNA before pol ε or pol δ takes over? How many pol ε and pol δ molecules are associated with individual replisomes? How often do poymerases exchange at the fork while synthesising the leading and lagging strands? To address these questions, we aim to visualise individual molecules in real time during replication of stretched DNA molecules. Our work will also provide important insight into how the replication machinery acts upon encountering different types of DNA damage.