A trimeric DNA polymerase complex increases the native replication processivity.

Abstract:

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase-polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Source / Purification, Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Structure and Structure/Function, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
SsoDpo1 Mikheikin AL2009 Molecular Weight 1.01E+05 Dalton Technique: Gel Filtration
SsoDpo1 Mikheikin AL2009 Cloned or native Cloned in E. coli
SsoDpo1 Mikheikin AL2009 Full length or truncated Full length
SsoDpo1 Mikheikin AL2009 Processivity 942bp
SsoDpo1 Mikheikin AL2009 Vmax 422 /second
SsoDpo1 Mikheikin AL2009 Kd 1.2uM
SsoDpo1 Mikheikin AL2009 Kd 542nM

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