Mapping Functional Substrate–Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs

Abstract

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5'-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5'-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme's active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP's 5'-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site.

Revealing an Internal Stabilization Deficiency in the DNA Polymerase β K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography

“…In general, the ternary complex crystal structures of pol β with the various dNTP analogues were found to be similar to the reference structure with a normal incoming dNTP. This observation facilitated interpretations regarding the acidity modification effects on the nucleotidyl transferase reaction versus other influences such as conformational effects . Interestingly, an exception to the structural conservation was found with one type of incoming dNTP analogue, the dATP analogue with the CF 2 group substitution.…”

“…This observation facilitated interpretations regarding the acidity modification effects on the nucleotidyl transferase reaction versus other influences such as conformational effects. 23 Interestingly, an exception to the structural conservation was found with one type of incoming dNTP analogue, the dATP analogue with the CF 2 group substitution. In this case, the CF 2 group appeared to introduce strain into the structure preventing the open to closed conformational change and distorting the position of α-helix N in the active site.…”

“…In this case, the CF 2 group appeared to introduce strain into the structure preventing the open to closed conformational change and distorting the position of α-helix N in the active site. 23 In the work presented here, we evaluated the possibility that the change at residue 289 from lysine to methionine, removing a stabilizing contact in a C-terminal region network of the enzyme, might destabilize the structure of this region and in turn alter the active site, as well. Because K289 is located in a surface region of the enzyme distal to the active site, one might expect the modification at this residue to have little impact on functions many angstroms away in the active site.…”

“…We previously reported a series of structures of wild-type pol β in a ternary complex with incoming dNTP β,γ-bisphosphonate analogues in which the oxygen atom bridging the β- and γ-phosphate groups has been replaced by a methylene group (CXY) − These analogues mimic dNTP as pol β substrates, but the leaving group is the corresponding bisphosphonate pCXYp instead of PP i . By appropriately choosing electron-donating (e.g., X, Y = CH 3 ) or electron-withdrawing (e.g., X, Y = F) substituents on the methylene carbon atom, one can significantly modulate its electronegativity.…”

Engineering-driven biological insights into DNA polymerase mechanism

“…Polymerase structures also have significant value in the engineering process, particularly from related structures harbouring a range of substrates, sampling mismatches [47,48], unnatural bases [49] and triphosphate analogues [50]. Those "families" of structures give a very detailed knowledge of spatial constraints that can affect polymerase function.…”