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Updated: Dec 6, 2025
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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
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The E3 ligase RFWD3 stabilizes ORC in a p53-dependent manner
Rosaline Y C Hsu1, Sumanprava Giri1, Yating Wang1
1Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign , Urbana, IL, USA.
Cell Cycle (Georgetown, Tex.)
|October 12, 2020
Summary
RFWD3, an E3 ubiquitin ligase, stabilizes the Origin Recognition Complex (ORC) and ORC-Associated protein (ORCA). This interaction is crucial for initiating DNA replication and ensuring efficient pre-replicative complex assembly.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- RFWD3 (Ring Finger and WD Repeat Domain 3) is an E3 ubiquitin ligase involved in DNA damage response and replication.
- Previous work showed RFWD3 stabilizes at replication forks, ubiquitylates RPA, and promotes replication progression.
Purpose of the Study:
- To investigate the role of RFWD3 in the initiation of DNA replication.
- To explore the interaction between RFWD3 and components of the pre-replicative complex.
Main Methods:
- Co-immunoprecipitation assays to detect protein interactions.
- Western blotting to assess protein levels.
- Depletion studies using siRNA to evaluate protein stability.
- Assessment of RFWD3's catalytic activity.
Main Results:
- RFWD3 was found to associate with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA).
- Overexpression of ORC/ORCA stabilized RFWD3, while RFWD3 depletion reduced ORC/ORCA levels, particularly in wild-type p53 cells.
- The catalytic activity of RFWD3 is essential for stabilizing ORC.
Conclusions:
- RFWD3 promotes the stability of ORC and ORCA.
- This stabilization by RFWD3 is critical for efficient pre-replicative complex (pre-RC) assembly and the initiation of DNA replication.
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