Structural Insights into Complete Metal Ion Coordination from Ternary Complexes of B Family RB69 DNA Polymerase.

Abstract:

We have captured a pre-insertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a non-hydrolyzable 2'-deoxyuridine 5'-,-substituted trisphosphate, dUpXpp, where X is either NH or CH2, opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca2+ and other four structures with catalytically competent Mn2+ or Mg2+. These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the -phosphorus atom (P) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the non-bridging Sp oxygen of the dNTP's -phosphate. The coordination bond length of metal-ion A as well as its ionic radius determines how close ptO3' can approach P. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to P enabling pyrophosphorolysis as well as assisting in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal-ion A coordination octahedron. When ptO3' is placed into the vertex of a non-distorted, idealized metal-ion A octahedron, it is within bond formation distance to P. This geometric relationship appears to be conserved among DNA polymerases of known structure.

Polymerases:

Topics:

Structure and Structure/Function

Status:

new topics/pols set partial results complete validated

Results:

No results available for this paper.

Entry validated by:

Structures:

3SPY
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.