Molecular mechanisms of DNA replication in mitosis

University Of Colorado Denver, Aurora CO

Investigators

Abstract

SUMMARY Each cell cycle, the genome must be faithfully duplicated and then divided appropriately into two progeny cells. Most of the genome is replicated in S phase of the cell cycle, and the mechanisms of DNA replication are well-characterized. However, cells sometimes have not finished replicating the entire genome as they enter mitosis, the phase of the cell cycle when chromosomes are segregated eventually leading to cell division. Instead, these remaining regions are replicated in mitosis in a likely “last-ditch effort” to produce separable chromosomes: cells without it face aneuploidy or mitotic catastrophe. Despite the importance of mitotic DNA replication, the mechanisms governing it are poorly understood. To uncover the molecular mechanisms of mitotic DNA replication, our research program will use the biochemical reconstitution of DNA replication using purified budding yeast proteins, which is ideally suited for addressing direct mechanistic questions. We will complement this with yeast genetics approaches, known for their significant contributions to our understanding of the cell cycle and DNA replication. Additionally, we have developed innovative strategies that combine reconstituted replication forks with yeast cell extracts to discover novel factors and explore the regulated timing of mitotic DNA replication. Our model of mitotic DNA replication is a multi-step process starting with the regulated inhibition and dismantling of the S phase replication machinery present at the replication fork to present a different structure and make space for mitotic proteins. This dismantling is followed by regulated unwinding and cleavage events, ultimately leading to a break-induced replication mechanism of strand-invasion, synthesis, and resolution. Our novel and innovative approaches will test this complex model and determine the coordination of these steps with other mitotic events occurring in distinct mitotic phases. We will also explore how and when mitotic DNA replication particularly occurs at hard-to-replicate regions of the genome like chromosome fragile sites and telomeres. S phase replication and the proteins so far implicated in mitotic DNA replication are well-conserved, so the mechanisms we uncover using the yeast system will provide a mechanistic framework that we will explore in other contexts in the future. We are interested in how genomic factors such as chromatin state and the presence of DNA damage or transcriptional conflicts play a role in mitotic DNA replication. Additionally, we seek to understand how diverse cellular contexts like developmental stage or disease state result in variable reliance on mitotic DNA replication, ultimately contributing to genome stability and cell viability.