Exploring the interplay between DNA replication kinetics and macromolecular protein assembly at the centromere

DNA replication is essential for the growth and proliferation of all living organisms. During DNA replication of chromosomes, centromeric regions must be replicated to enable chromosome organization and cell division. Centromeres contain specialized DNA sequence of a chromosome, capable of binding multiple proteins, that links a pair of sister chromatids. Currently, little is known regarding the interplay between the DNA replication machinery and centromere activation in cells. During the research grant we combined a variety of research approaches including genetics, cell biology, live-cell microscopy, and computational methods to study replication fork progression through centromeric region and understand replication stress. To study replication through centromeric regions and understand the relationships between replication and active transcription at centromeric site, we further developed our live-cell imaging approach for the simultaneous monitoring of replication and transcription at the same locus. Using this approach, we studied transcription-replication conflicts in real-time (1) and laid the foundation for examination of replication through active centromeres. In parallel, we examined the behavior of chromosomes with two centromeres on the same DNA strand (dicentric chromosome) to study the natural cellular forces and repair pathways required for DNA breakage and conversion to monocentric derivative chromosomes. Through the analysis of dicentric chromosome behavior, the dependency on DNA repair machinery, and direct observation of cross-overs using single- nucleotide polymorphisms between the two centromeres, we found that replication pausing is the most likely initiating event leading to genetic recombination between the centromeres (2). Finally, we developed an approach for enhanced imaging of fluorescent proteins in live yeast cells by the binding of single chain fragment variable (scFv)-fluorescent protein fusion to multiple HA-tagged proteins. This approach enables highly sensitive DNA-damage detection, the facile labelling of sensitive proteins, and the enhanced detection of proteins localized to different yeast cellular organelles (3).