Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

Abstract:

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Polymerases:

Topics:

Kinetic Parameters, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Phi29 de Vega M1999 Cloned or native Cloned in E. coli
Phi29 D12AD66A de Vega M1999 3-5' Exonuclease (proofreading) No
Phi29 D12AD66A de Vega M1999 Cloned or native Cloned in E. coli
Phi29 D12AD66A de Vega M1999 Tagged Unspecified
Phi29 D12AD66A de Vega M1999 Full length or truncated Full length
T7 de Vega M1999 3-5' Exonuclease (proofreading) Yes
Phi29 de Vega M1999 3-5' Exonuclease (proofreading) Yes
Phi29 de Vega M1999 5-3' Exonuclease Unspecified
Phi29 de Vega M1999 Full length or truncated Full length
Phi29 de Vega M1999 Vmax 500 /second Reaction: 3-5' Exonuclease; Substrate: DNA template

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.