Research Project
8114138
DESCRIPTION (provided by applicant): Replication of genomic DMA involves the coordinated activity of a large number of proteins. The replisome, the molecular machinery of DMA replication, unwinds the double-stranded DMA, provides primers to initiate synthesis, and polymerizes nucleotides onto each of the two growing strands. Remarkable progress has been made in characterizing the structural and functional properties of the individual components;their coordination at the replication fork is less well understood. The dynamic nature of the replisome makes it difficult to probe these processes with ensemble-averaging techniques. We propose to use single molecule techniques to study the mechanisms that coordinate replication in fully assembled and functional replication complexes. We will use in vitro reconstituted bacteriophage T7 replisomes as a model system and study the kinetics of both leading- and lagging-strand synthesis on a single-molecule level. In particular, we will determine how the continuous polymerization at the leading-strand is coupled to the frequently interrupted lagging-strand synthesis and what enzymatic step triggers the recycling of the lagging-strand DMA polymerase. Furthermore, we will use single-molecule fluorescence resonance energy transfer between labeled components of the replisome during replication to determine how the large conformational changes required for lagging-strand DNA polymerase recycling are facilitated. Public Health relevance: The ability of DNA replication proteins to quickly and accurately replicate DNA without stalling or dissociating is crucial to maintain genomic stability and thus to prevent cancer. By observing individual replication complexes and recording "molecular movies" of their enzymatic activities, we will provide new insights into the general mechanisms underlying the coordination of proteins at the replication fork. Furthermore, the novel single-molecule methodologies we propose to develop will be directly applicable to studying the dynamics of other, more complex systems with unprecedented detail.
