Observing protein dynamics during DNA-lesion bypass by the replisome.

Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example of a roadblock to replication is chemical modifications in the DNA caused by exposure to ultraviolet light. Protein dynamics are thought to play a crucial role to the molecular pathways that occur in the presence of such DNA lesions, including potential damage bypass. Therefore, many assays have been developed to study these dynamics. In this review, we discuss three methods that can be used to study protein dynamics during replisome-lesion encounters in replication reactions reconstituted from purified proteins. Specifically, we focus on ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy. We discuss two key model DNA replication systems, derived from