Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69.

Abstract:

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.

Polymerases:

Topics:

Accessory Proteins/Complexes, Kinetic Parameters

One line summary:

The presence of the RB69 DNA polymerase at the primer terminus results in a 57-fold increase in gp32 (SSB) affinity for ssDNA as a result of DNA polymerase:SSB cooperativity.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
RB69 D222AD327A Sun S2003 Kd 124nM Reaction: Polymerase-DNA interaction; Substrate: DNA template; Technique: fluorescence titration
RB69 D222AD327A Sun S2003 Kd 353nM Reaction: Polymerase-DNA interaction; Substrate: DNA template; Technique: fluorescence titration (gp32 added)

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.