Optimal and Variant Metal-Ion Routes in DNA Polymerase β’s Conformational Pathways

Abstract

To interpret recent structures of the R283K mutant of human DNA repair enzyme DNA polymerase β (pol β) differing in the number of Mg(2+) ions, we apply transition path sampling (TPS) to assess the effect of differing ion placement on the transition from the open one-metal to the closed two-metal state. We find that the closing pathway depends on the initial ion position, both in terms of the individual transition states and associated energies. The energy barrier of the conformational pathway varies from 25 to 58 kJ/mol, compared to the conformational energy barrier of 42 kJ/mol for the wild-type pol β reported previously. Moreover, we find a preferred ion route located in the center of the enzyme, parallel to the DNA. Within this route, the conformational pathway is similar to that of the overall open to closed transition of pol β, but outside it, especially when the ion starts near active site residues Arg258 and Asp190, the conformational pathway diverges significantly. Our findings should apply generally to pol β, since R283K is relatively far from the active site; further experimental and computational work are required to confirm this. Our studies also underscore the common feature that less active mutants have less stable closed states than their open states, in marked contrast to the wild-type enzyme, where the closed state is significantly more stable than the open form.

Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β

“…Interestingly, Elber and coworkers 52 determined a single TS from the minimum free energy path between the open and closed states of DNA Pol in contrast to the several metastable states proposed by Ref. 41, suggesting that there was no evidence favoring the more complex model. 52 The above issues become more critical when one tries to explore the catalytic role of the metals (see Results section).…”

“…For example Refs. concluded that the Mg path plays a major role in catalysis. This overlooked the fact(s) that what counts is the overall free energy along the least energy path rather than artificial fluctuating barriers; confusing the obvious fact that the potential energy changes drastically upon moving the catalytic metals (see Ref.…”

New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

“…Binding of the catalytic metal alters the sugar pucker (3′-endo) of the primer terminus repositioning O3′ for in-line attach on the α-phosphate of the incoming nucleotide [5, 19]. Computational studies also indicate that the path to its binding site can influence the open/closed subdomain transition prior to chemistry [20]. …”

Insertion of oxidized nucleotide triggers rapid DNA polymerase opening

“…The free energy barriers of Mg(p) and Mg(n) dissociations from each system are ∼12–13 k B T and ∼10–11 k B T, respectively (Table 2 ). These free energy patterns of metal ion dissociations during the opening pathway after chemistry are opposite those in the closing pathway of the pol-β before chemistry ( 68 ).…”

“…The appropriate order parameters for TPS simulations are chosen from the crystallographic data ( 16 , 41 ), molecular dynamics ( 17 , 18 ) and prior TPS studies ( 45 , 65 – 68 ). These works have shown that key active site residues (Asp190, Asp192, Arg258 and Phe272) and metal ions motions serve as measures of pol-β closing pathway before chemistry.…”