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Human Kaposi's sarcoma herpesvirus processivity factor-8 functions as a dimer in DNA synthesis
Xulin Chen1, Kai Lin, Robert P Ricciardi
1Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
The Journal of Biological Chemistry
|April 13, 2004
Summary
Kaposi's sarcoma herpesvirus DNA polymerase (Pol-8) requires the processivity factor (PF-8) for extensive DNA synthesis. PF-8 functions as a dimer, with specific regions essential for dimerization and Pol-8 stabilization.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Kaposi's sarcoma herpesvirus DNA polymerase (Pol-8) exhibits limited processivity independently.
- Herpesvirus processivity factors, unlike eukaryotic sliding clamps, do not require ATP or clamp loaders for DNA binding.
Purpose of the Study:
- To elucidate the mechanism by which the Kaposi's sarcoma herpesvirus processivity factor (PF-8) enhances Pol-8 processivity.
- To investigate the structural requirements for PF-8 dimerization and its role in Pol-8 function.
Main Methods:
- Purification of PF-8.
- Dimerization studies in solution and on DNA.
- Mutational analysis of PF-8 (396R).
- Assessment of Pol-8 activity in the presence of wild-type and mutant PF-8.
Main Results:
- PF-8 was purified and shown to be a dimer in solution and on DNA.
- Specific regions of PF-8 (residues 277-304 and N-terminal 21 amino acids) are critical for dimerization.
- PF-8 dimerization correlates with Pol-8 binding and stabilization on primer-template DNA.
- Truncation of the PF-8 C-terminus (26 residues) abolished long-chain DNA synthesis despite maintaining dimerization and Pol-8 binding.
Conclusions:
- PF-8 functions as a dimer to confer processivity to Pol-8.
- Full-length PF-8 is essential for Pol-8 to synthesize thousands of nucleotides.
- The dimeric structure of PF-8 is crucial for its function in viral DNA replication.