DNA replication termination involves the meeting of two converging replication forks, completion of DNA synthesis, and the unloading of replisome proteins (Dewar et al., Nature, 2015). Unloading of the CDC45- MCM2-7-GINS (CMG) replicative helicase involves ubiquitylation of the MCM7 subunit by a cullin RING family ubiquitin ligase (CRL) and activity of the AAA-ATPase, p97 (Maric et al., Science, 2014; Moreno et al., Science, 2014). However, a detailed understanding of how ubiquitylation regulates CMG unloading during replication termination has remained elusive. The identity of the CRL and the ubiquitylated residues on its substrate remain unknown. Additionally, the trigger for CRL recruitment and MCM7 ubiquitylation has not yet been characterized. We favor a model where the encounter of the CMG replicative helicase with double- stranded DNA triggers ubiquitylation of the MCM7 subunit, which acts as the signal to recruit the p97 AAA- ATPase to disassemble CMG and associated replisome proteins from chromatin. I propose to use the cell- and nucleus-free Xenopus egg extract system to investigate the mechanism of ubiquitylation during DNA replication termination. A postdoctoral fellow in the Walter lab has performed a mass spectrometry screen and identified several candidate proteins involved in replication termination, including several ubiquitin signaling proteins. From this pool of identified proteins, I will identify the ubiquitin ligase (UBL) involved in ubiquitylating CMG by depleting candidate UBLs from egg extracts and determining the effect on CMG unloading. I will also identify the ubiquitylated residues on MCM7 using mass spectrometry, and determine if they are functionally important for unloading of CMG and associated replisome components. Lastly, I will investigate whether the passage of double-stranded DNA through the central channel of the CMG helicase is the molecular event that triggers ubiquitylation. I will do this by loading CMG onto single-stranded and double-stranded oligonucleotides, and determining whether CMG is unloaded from double-stranded DNA upon exposure to egg extracts. Together, this project will elucidate the mechanism of how ubiquitylation and CRLs regulate CMG unloading during replication termination. Characterization of whether and how these proteins are involved in CMG helicase unloading will illuminate how the fundamental process of replication termination occurs. Furthermore, understanding the mechanism of ubiquitylation in replication termination will enhance our understanding of the diverse functions of UBLs in cells, shed light on how their improper function leads to disease, and possibly provide insights into new treatments.

Public Health Relevance

This project will investigate how the cellular machinery responsible for replicating genomic DNA is removed after DNA replication has been completed, and how this removal is regulated to prevent harm to the cell. Understanding how this removal is controlled will provide insight into how improper function and regulation of this cellular machinery leads to disease, including neurodegenerative diseases and cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Sledjeski, Darren D
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Harvard Medical School
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Dewar, James M; Low, Emily; Mann, Matthias et al. (2017) CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination. Genes Dev 31:275-290