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Cofractionation of HeLa cell replication proteins with ors-binding activity
M T Ruiz1, C E Pearson, T Nielsen
1McGill Cancer Centre, Department of Medicine, McGill University, Montréal, Québec, Canada.
Journal of Cellular Biochemistry
|June 1, 1995
Summary
Researchers identified a specific protein fraction that binds to a minimal DNA sequence (ors 8) and supports its replication. This protein fraction contains key replication factors, suggesting its role in DNA origin function.
Area of Science:
- Molecular Biology
- Mammalian DNA Replication
Background:
- Origin-enriched sequences (ors) are crucial for DNA replication initiation.
- A specific 186 bp fragment of ors 8 is essential for its replication activity.
Purpose of the Study:
- To identify proteins interacting with the minimal ors 8 DNA sequence.
- To characterize the protein fraction responsible for ors 8 replication.
Main Methods:
- Fractionation of HeLa cell extracts using DEAE-Sephadex and Affi-Gel Heparin chromatography.
- In vitro replication assays using ors 8 plasmid.
- Glycerol gradient sedimentation and band-shift elution experiments to identify binding proteins.
Main Results:
- A specific protein fraction was isolated that binds to the 186 bp ors 8 fragment and supports its in vitro replication.
- The ors binding activity (OBA) in this fraction has an estimated molecular weight of approximately 150 kDa.
- Band-shift analysis identified two predominant proteins of 146 kDa and 154 kDa within the OBA fraction.
- The OBA fraction is enriched in known replication factors including polymerases alpha and delta, topoisomerase II, and replication protein A (RP-A).
Conclusions:
- A specific protein fraction containing ors binding activity (OBA) is essential for the replication of ors 8.
- The identified OBA is likely composed of proteins around 146-154 kDa and includes key replication machinery components.
- This study elucidates a critical protein-DNA interaction in mammalian DNA replication initiation.