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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
A role for USP7 in DNA replication
Madhav Jagannathan1, Tin Nguyen, David Gallo
1Department of Molecular Genetics, University of Toronto, Toronto, Canada.
Abstract:
The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.
Insights
The ubiquitin-specific protease USP7, working with MCM-BP, helps unload minichromosome maintenance (MCM) complexes from chromatin after DNA replication. Its absence slows DNA synthesis by preventing MCM complex dissociation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The minichromosome maintenance (MCM) complex is crucial for DNA replication, loading onto chromatin post-mitosis and unloading post-S phase.
- The unloading mechanism of MCM complexes from chromatin is not fully understood, with MCM binding protein (MCM-BP) implicated.
Purpose of the Study:
- To investigate the role of USP7 in the unloading of MCM complexes from chromatin during DNA replication.
- To elucidate the interaction between MCM-BP and USP7 and its functional significance.
Main Methods:
- Biochemical assays and structural analyses to characterize the USP7-MCM-BP interaction.
- USP7 knockout studies to assess effects on DNA replication.
- Chromatin immunoprecipitation and protein level analysis to examine MCM protein dynamics.
Main Results:
- MCM-BP directly interacts with USP7 on chromatin, tethering USP7 to MCM proteins via the (155)PSTS(158) sequence.
- USP7 knockout leads to slowed progression through late S phase without affecting fork rate or origin usage.
- Lack of USP7 causes increased MCM protein levels on chromatin due to impaired dissociation in mid- to late S phase.
Conclusions:
- USP7, in conjunction with MCM-BP, plays a critical role in the unloading of MCM complexes from chromatin at the end of S phase.
- USP7 is essential for efficient DNA replication progression by facilitating MCM complex removal from chromatin.
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