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The PCNA from Thermococcus fumicolans functionally interacts with DNA polymerase delta
G Henneke1, J P Raffin, E Ferrari
1Ifremer, Plouzané, 29280, France.
Abstract:
We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated M(r) of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta). When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta. Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals.
Insights
We cloned the gene for proliferating cell nuclear antigen (PCNA) from the hyperthermophilic archaeon Thermococcus fumicolans. This archaeal PCNA efficiently stimulated mammalian DNA polymerase delta, suggesting functional conservation across domains of life.
Area of Science:
- Molecular Biology
- Biochemistry
- Archaea Biology
Background:
- Proliferating cell nuclear antigen (PCNA) is a crucial protein involved in DNA replication and repair in eukaryotes.
- Understanding PCNA's function in archaea can provide insights into its evolutionary conservation and functional mechanisms.
Purpose of the Study:
- To clone and characterize the gene encoding PCNA from the hyperthermophilic archaeon Thermococcus fumicolans (Tfu).
- To investigate the functional interaction of Tfu PCNA with both homologous and heterologous DNA polymerases.
Main Methods:
- Gene cloning and overexpression of Tfu PCNA in Escherichia coli.
- Purification of recombinant Tfu PCNA.
- In vitro assays testing Tfu PCNA's effect on Tfu DNA polymerase I and calf thymus DNA polymerase delta activity.
Main Results:
- The Tfu PCNA gene was successfully cloned and expressed, yielding a protein of approximately 33.5 kDa.
- Tfu PCNA showed limited stimulation of the homologous Tfu DNA polymerase I.
- Surprisingly, Tfu PCNA significantly enhanced the activity of calf thymus DNA polymerase delta, outperforming human PCNA in this assay.
Conclusions:
- PCNA function is conserved from hyperthermophilic archaea to mammals, indicating a deep evolutionary preservation of this essential replication factor.
- The differential stimulation of homologous versus heterologous polymerases suggests specific functional adaptations or conserved interaction interfaces of PCNA.