Understanding replication fork stalling at the single-molecule level inside bacterial cells


DNA replication is one of the most fundamental processes in cell biology (Fig. 1). For every round of cell division, the cell must synthesize an accurate copy of the genome. This is typically achieved with less than one mistake per 109 nucleotides copied, which is a testimony to the robustness of the replication machinery that [1].
However, there are situations when replication fork advancement is impaired: one speaks of fork stalling, which, when accompanied by loss of key components of the replication fork, results in fork collapse. Fork stalling may occur spontaneously during replication [2] or result from conflicts with other proteins, e.g. in replication-transcription collisions [3]. Stressful environmental conditions also pose significant challenges to replication fork advancement [4]. Failure to properly rescue stalled forks results in errors in the genome, which, when numerous, constitute genome instability and threaten cell viability.
The overarching goal of the proposed research is to understand the in vivo response to fork stalling. This includes the following milestones:

A. Description of the stoichiometry and dynamics of the replisome and restart proteins during fork stalling.
B. Observation of the roles of accessory helicases in the repair and restart of stalled forks.
C. Measurement of the switching behavior of translesion polymerases at replication forks.

To achieve these milestones, we will employ a multi-disciplinary approach that harnesses genetic engineering and molecular biology to enable live, high-resolution imaging of cellular replication in the bacterium Escherichia coli (E. coli), well known generally as an excellent model system for molecular- and microbiology, and in particular for mechanistic studies in vivo [5, 6].





Prof. dr. N.H. Dekker

Verbonden aan

Technische Universiteit Delft, Faculteit Technische Natuurwetenschappen, NanoScience - Kavli Institute of Nanoscience Delft


01/01/2018 tot 01/01/2021


€ 378.877