Browsing by Subject "genome instability"

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    Mcm10 And Rnf4 Protect The Integrity Of The Genome By Preventing Dna under-replication
    (2023-04) Oram, Marissa
    Cancer is one of the leading causes of death worldwide and despite major advancements, its intricate mechanisms continue to perplex scientists. One characteristic that is prevalent in almost all cancer types is genome instability, defined as structural chromosomal alterations. The integrity of the genome relies on faithful genome inheritance and a complex network of pathways has evolved to monitor the progression of active replication forks. When replication forks stall, they trigger ‘replication stress’, a major cause of genome instability. My dissertation research focused on deepening our understanding of the pathways activated in response to chronic replication stress that help promote cancer cell survival. Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, and mitosis, summarized in Chapter 1 of this dissertation. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Notably, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. While the role of the mammalian STUbL, RING finger protein 4 (RNF4), has been well-documented in DNA double-strand break (DSB) repair (reviewed in Chapter 1), its impact during replication stress remains elusive. Interestingly, yeast STUbL mutants are not sensitive to transient exposure of a replication inhibitor but prolonged treatment significantly reduces cell viability1. We hypothesized that mammalian RNF4 is engaged in managing chronic replication stress to promote cell survival. To induce replication stress during each cell cycle, we used a genetic approach in which we mutated an essential replication factor, minichromosome maintenance protein 10 (MCM10), required for both replication initiation and DNA elongation. Chapter 2 of this dissertation describes the pathways MCM10 and RNF4 participate in to prevent severe DNA under-replication. We discovered a role for RNF4 in origin firing that is independent of the function of MCM10. We conclude that RNF4 prevents DNA under-replication under conditions of MCM10 deficiency by maintaining a critical threshold level of origin activation. To date, our molecular understanding of chronic replication stress in cancer remains limited. This gap in knowledge is partly due to the astonishing diversity of genomic mutations and structural variations found in human tumors, highlighting the necessity for a better mechanistic understanding of genome instability. Replication stress is the primary endogenous source of genomic instability, yet it is well-tolerated by cancer cells, and replication stress tolerance mechanisms are potentially key to chemoresistance. This is discussed in Chapter 3. My work argues that RNF4, which is commonly upregulated in epithelial malagnancies2, promotes replication stress tolerance and cancer cell survival. We propose that exacerbating DNA under-replication by targeting RNF4 will induce growth arrest or cell death in highly proliferative colon cancer cells, potentially limiting genetically acquired drug resistance.