“Gate-keeper” Residues and Active-Site Rearrangements in DNA Polymerase μ Help Discriminate Non-cognate Nucleotides

Abstract

Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these “gate-keeper” residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP<A(syn):dATP<T:dCTP<A:dGTP<A(syn):dGTP<A:dCTP<A:dATP. This sequence agrees with available kinetic data for non-cognate nucleotide insertions, with the exception of A:dGTP, which may be more sensitive to the template sequence. The structures and conformational aspects predicted here are experimentally testable.

Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair

“…Below the nascent ddCTP‐G base pair, the side chains of R454 and R458 become more ordered and change rotamers compared to previously known Tdt structures, blocking one side of the nascent base pair (Figs D and B). This is consistent with the role of these two conserved side chains for catalysis that has been underlined by Molecular Dynamics simulations (Li & Schlick, ). These side chains, along with L398 and F405, are literally isolating two base pairs (micro‐homology and nascent) from the rest of the DNA substrate.…”

“…This is likely due to the slightly different SD1 and SD2 regions/motifs in the two proteins (colored magenta and orange, respectively). (Li & Schlick, 2013, 2010. One possible scenario, which is compatible with a number of Pol mu mutants that have a 3 0 -5 0 -exonuclease phenotype (Rosario et al, unpublished), would be a sequential effect and active role of Loop1 in checking the MH-bp in Pol mu-that would not exist in Tdt.…”

“…The role of Mn 2+ (or other divalent transition metal ions) also needs to be investigated in detail, especially their possible role in polarizing nearby water molecules. Based on molecular dynamics simulations, other authors have postulated the existence of an intermediate state (check point) in the reaction path for Pol mu (Li & Schlick, , ). One possible scenario, which is compatible with a number of Pol mu mutants that have a 3′–5′‐exonuclease phenotype (Rosario et al , unpublished), would be a sequential effect and active role of Loop1 in checking the MH‐bp in Pol mu—that would not exist in Tdt.…”

Ribonucleotide discrimination by translesion synthesis DNA polymerases

“…Molecular dynamics simulations (Li and Schlick, 2013) have shown that compared to pols β and λ, pol μ has much more flexible active site. These findings provide an additional explanation for the unique ability of pol μ to accommodate ribonucleotides in the binding cleft and to catalyze their insertion.…”

Analysis of β-Structure Interfaces of Various Protein Folds

“…In contrast, though an average of 80-89% of residues contributes to form hydrogen bonds in ferredoxin like, double stranded beta helix, avidin/streptavidin, tautomerase/MIF, UBC folds structures, only 10-15% of the interface residues contribute to the intermolecular hydrogen bonds, indicating that the interface is not extensive and contact area is confined to specific locations; in most of the folds the residues in these locations involved in the edge strand gate keeping role and any perturbation can distort the dimer interface. 66 12.0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48 3.0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48 120.0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 interactions and emphasizes importance of considering protein fold during the designing the -interfaces.…”