A novel 4-reporter system for measuring DNA replication in yeast using live-cell imaging

The replication of chromosomes is a highly complex process, carried out accurately and efficiently during each round of the cell cycle by a large protein complex known as the replisome. The replisome must progress along the chromosome at high speed while overcoming various obstacles, such as secondary structures, highly transcribed regions, and damaged DNA. Studying replication mechanistically requires efficient methodologies for assaying replisome function in living cells under various conditions. We have developed a novel experimental system for directly measuring the progression of single replisomes in living cells, by monitoring two fluorescently labeled genomic loci downstream from an origin of replication. The system relies on arrays of bacterial operator sequences, lacO and tetO, which are integrated into the genome and bound by the cognate repressors fused to fluorescent proteins. This system has been successfully employed to study the factors that enable rapid replisome progression, and the causes of replisome stalling at obstacles. However, the existing system can only track replication over a single interval. Here, we expand the system by developing two novel operator arrays: a mutant version of the tetO array bound by an orthogonal mutant repressor, and an array of bacterial sequences bound by the LexA repressor. These new arrays, coupled with optimized 4-color live-cell imaging, enable tracking of four genomic loci simultaneously. They could be employed for measuring replication rates over different chromosomal spans, enabling the study of changes in replisome progression before, during and after encountering obstacles.