Temporal regulation of DNA replication at the G1/S transition of the cell cycle
- A fundamental challenge in cell division is replicating genomic DNA once and only once each cell cycle, such that daughter cells faithfully inherit their genome. In eukaryotes, duplicating DNA precisely once is critically dependent on separating DNA replication into two distinct stages: "origin licensing" during G1 phase, and "origin firing" during S phase. During origin licensing, cells demarcate future sites of DNA synthesis by loading inactive MCM helicases onto origins of replication. At the start of S phase, cells begin origin firing, whereby replication factors are recruited to the inactive helicases to form the active CMG helicases and replication forks that duplicate DNA. Critically, licensing is inhibited once DNA synthesis starts, such that newly synthesized DNA cannot be re-licensed. Failure to coordinate these processes can lead to re-replication of DNA, in which DNA is copied more than once within a single cell cycle, causing DNA damage and genome instability and ultimately can produce the mutations underlying diseases such as cancer. This dissertation describes work done to understand the regulation and signaling which coordinates G1 phase origin licensing and S phase origin firing. In human cells, inhibition of licensing in S phase centers around inhibition of licensing factor CDT1 starting from S phase entry. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Using quantitative microscopy and in vitro reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase progression at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis. .
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|Ratnayeke, Nalin Joseph
|Ferrell, James Ellsworth
|Ferrell, James Ellsworth
|Degree committee member
|Stanford University, School of Medicine
|Stanford University, Department of Chemical and Systems Biology
|Statement of responsibility
|Submitted to the Department of Chemical and Systems Biology.
|Thesis Ph.D. Stanford University 2023.
- © 2023 by Nalin Joseph Ratnayeke
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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