ESSENTIAL FUNCTIONS OF THE SMC5/6 COMPLEX IN MAMMALIAN DNA REPLICATION
Johns Hopkins University
Structural maintenance of chromosome (SMC) complexes are essential effectors of chromosome homeostasis conserved through all domains of life. There are three classes of SMC complexes expressed in all eukaryotes – cohesin, condensin, and SMC5/6. Cohesin and condensin possess well-defined core functions in driving changes to chromosome superstructure to promote sister chromatid cohesion and chromosome condensation. Here, we focus on the less characterized SMC5/6 complex, which has consistently been implicated in maintenance of replication fork integrity. Mutations in subunits of the SMC5/6 complex are linked to human diseases, ranging from developmental abnormalities to malignancies. The research presented in this thesis focuses on the essential role of the SMC5/6 complex during mammalian DNA replication, disruption of which contributes to developmental defects in mammals and lethality in all proliferative cell types. In Chapter I, we discuss current knowledge of the structure, biochemical properties, and replication fork-associated functions of SMC5/6, and how these functions ensure organism development and homeostasis. SMC5/6 is required for embryonic development. In the following chapters, we describe the development of two systems to study the functions of SMC5/6 during embryonic development – a Smc5 conditional knockout (cKO) mouse, and mouse embryonic stem cells (mESCs) harboring the auxin-inducible degron (AID) system. The AID system enables direct, rapid, and reversible target protein degradation, allowing for fine-tuned control of protein levels in various cellular contexts. In Chapter II, we describe the integration of the AID system in mESCs for modeling SMC5/6 deficiency in vitro. In Chapter III, we describe neurodevelopmental defects in mice that ensue due to cKO of Smc5. We find that Smc5 cKO mice exhibit neurodevelopmental defects due to neural progenitor cell apoptosis, leading to a reduction in cortical layer neurons. Using Smc5 cKO and auxin-induced depletion of SMC5, we determine that SMC5/6 deficiency triggers a CHEK2- and p53-dependent DNA damage response. In addition, we find that depletion of SMC5/6 components leads to overall replication fork instability and incomplete DNA replication prior to mitosis in stem cells. In Chapter IV, we characterize replication fork dynamics in detail with acute auxin-mediated depletion of SMC5 in the contexts of replication fork stall and restart. We determine that SMC5/6 is required for replication fork restart and describe a mechanism of SMC5/6 action during replication stress, which promotes recruitment of key stabilizing factors to the replication fork. Overall, the findings from this thesis provide new insights into how the SMC5/6 complex coordinates the replication stress response to maintain the integrity of stalled replication forks.
SMC5/6, DNA repair, genome integrity, DNA replication