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OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication
Molecular Biology of the Cell
|March 9, 1999
Summary
This study identifies a specific DNA sequence that binds to ors-binding activity (OBA). This binding protein is identified as human Ku antigen, implicating Ku in mammalian DNA replication.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Ors-binding activity (OBA) was previously purified from HeLa cells.
- OBA specifically interacts with the ors8 minimal replication origin and supports in vitro replication.
Purpose of the Study:
- To identify the specific DNA region responsible for OBA binding.
- To identify the protein component of OBA involved in DNA binding.
- To investigate the role of OBA in mammalian DNA replication.
Main Methods:
- Competition band-shift analyses using DNA subfragments.
- Band-shift elution and Southwestern analysis.
- Microsequencing and affinity purification.
- In vitro DNA replication assays.
Main Results:
- A 59-bp fragment of the ors8 origin competed efficiently for OBA binding.
- A 36-bp sequence (A3/4) within this fragment showed high affinity for OBA.
- The 92-kDa polypeptide in OBA was identified as the 86-kDa subunit of human Ku antigen.
- OBA fractions contained Ku subunits and DNA-dependent protein kinase catalytic subunit.
- Ku was implicated in mammalian DNA replication through in vitro experiments.
Conclusions:
- Human Ku antigen is a key component of OBA.
- Ku plays a role in mammalian DNA replication initiation or progression.