In cells, replication is thought to start at fixed genomic locations, termed origins, that function both as sites where replisome assembly begins and the process of chromosome duplication takes place. We reconstituted the process using purified proteins and studied challenges by other machineries. We discovered that RNA polymerase, which is responsible for gene expression, can push replication factors to new locations where they continue replisome assembly (Scherr et al. Cell Reports 2022). This observation calls for a major revision of the classic model of DNA replication in which the location of replisome assembly is the same location where chromosome duplication starts. Building on this success, we next investigated challenges from cohesin, a machine that creates loops in chromosomes. We discovered that replisome assembly is a barrier to cohesin movement that determines the three-dimensional organization of chromosomes in developing embryos (Dequeker et al. Nature 2022).
When chromosomes are copied, the two individual strands of the DNA double-helix must be separated so the replisome can gain access to the genomic information. However, this process can rapidly generate tangled DNA. To overcome this problem, cells rely on topoisomerases. We developed a novel instrument called Flow Magnetic Tweezers (FMT) that allows for massive parallel imaging of topoisomerase activity on individual DNA molecules. Using this technique, we unexpectedly discovered that drugs targeting topoisomerases, in this case Ciprofloxacin, lead to the formation of remarkably stable complexes that resist extreme forces and twisting (Agarwal et al. Nature Communications, 2020). These observations provide new insights into the mechanism of drug action revealing why these drugs are able to kill cells effectively.
During chromosome duplication, the replisome is frequently challenged by obstacles. Among these are nucleosomes that are the basic units of chromosome organization. Chaperone proteins are thought to help the replisome overcome these barriers, but the mechanism has remained unknown. We discovered that the chaperone FACT directly interacts with the replisome at a location near where nucleosomes are encountered (Safaric et al. Nucleic Acids Research 2022). We also discovered an interaction network in FACT that helps take apart nucleosomes. Taken together, these observations revealed how the units of chromosome organisation are processed during replication.
