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Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen
Padmini S Kedar1, Soon-Jong Kim, Anthony Robertson
1Laboratory of Structural Biology, NIEHS/National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
The Journal of Biological Chemistry
|June 14, 2002
Summary
Proliferating cell nuclear antigen (PCNA) interacts with DNA polymerases. A specific motif (PIM) on DNA polymerase beta is crucial for this interaction, essential for DNA replication and repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Proliferating cell nuclear antigen (PCNA) is vital for DNA replication and repair.
- PCNA interacts with numerous proteins, some containing a PCNA Interacting Motif (PIM).
Purpose of the Study:
- To investigate the interaction between PCNA and DNA polymerase beta (beta-pol).
- To determine if PIM-like sequences in beta-pol mediate this interaction.
Main Methods:
- Yeast two-hybrid analysis
- In vitro and in vivo complex formation assays
- Immunoprecipitation
- Overlay binding assays
- Competition assays with synthetic peptides
- Site-directed mutagenesis of beta-pol PIM mutant (H222A/F223A)
Main Results:
- Human and rat beta-pol possess three PIM-like regions.
- beta-pol and PCNA form a complex in vitro and in vivo.
- A PIM mutant (H222A/F223A) of beta-pol showed a blocked interaction with PCNA.
- Competition assays confirmed the PIM's importance in the interaction.
Conclusions:
- At least one PIM-like sequence in beta-pol functions as a PIM.
- This functional PIM is required for the interaction between beta-pol and PCNA.
- The findings elucidate a key molecular interaction in DNA metabolism.