Modeling DNA polymerase μ motions: subtle transitions before chemistry

Abstract

To investigate whether an open-to-closed transition before the chemical step and induced-fit mechanism exist in DNA polymerase μ (pol μ), we analyze a series of molecular-dynamics simulations with and without the incoming nucleotide in various forms, including mutant systems, based on pol μ's crystal ternary structure. Our simulations capture no significant large-scale motion in either the DNA or the protein domains of pol μ. However, subtle residue motions can be distinguished, specifically of His(329) and Asp(330) to assemble in pol μ's active site, and of Gln(440) and Glu(443) to help accommodate the incoming nucleotide. Mutant simulations capture a DNA frameshift pairing and indicate the importance of Arg(444) and Arg(447) in stacking with the DNA template, and of Arg(448) and Gln(440) in helping to stabilize the position of both the DNA template and the incoming nucleotide. Although limited sampling in the molecular-dynamics simulations cannot be ruled out, our studies suggest an absence of a large-scale motion in pol μ. Together with the known crystallization difficulties of capturing the open form of pol μ, our studies also raise the possibility that a well-defined open form may not exist. Moreover, we suggest that residues Arg(448) and Gln(440) may be crucial for preventing insertion frameshift errors in pol μ.

Figure 1

Our model of the murine pol μ ternary complex. (a) Protein domains, DNA strands, and Mg²⁺. (b) Starting loop conformations. Structure I (dark), from regular MD, used in simulations I, II, and VII–XVII; structure II (light), from 299 K REMD replica, used in simulations III and IV; structure III (medium), from 301 K REMD, used in simulations V and VI. Residues 360–420 of each structure are shown; other protein residues are drawn with a smaller bond radius. (c) Shifted-DNA model. DNA in the shifted-DNA model (dark) was taken from pol λ's binary structure, superimposed with DNA in pol μ's ternary structure (light). The protein is also shown as green. (d) Open-thumb model. The thumb region of pol μ (dark) was shifted to an open form comparable to pol β's binary structure and compared with pol μ's original ternary structure (light).

Figure 2

Protein and DNA motions in pol μ simulations. (a) Protein motions (left) and DNA motions (right) of pol μ in selected simulations (red, simulation I; green, simulation II; blue, simulation III; black, simulation V). For clarity, DNA bases other than A5 in simulations II, III, and V are not shown. (b) DNA position in simulation XIV (shifted-DNA model), before (brown) and after simulation (purple), compared with simulation II (green). (c) Protein conformation in simulation XV (open-thumb model), before (brown) and after (purple) simulation, compared with simulation II (green). The motion of α-helix N in the thumb is shown. (d) Protein motions (left) and DNA motions (right) of pol μ in mutant simulations compared with wild-type simulation I (red, simulation I; cyan, simulation XI; orange, simulation XII; purple, simulation XIII). DNA bases other than A5 in simulations XI, XII, and XIII are not shown. The shift of A5 base is indicated. (e) Interactions between DNA and residues Arg⁴⁴⁴, Arg⁴⁴⁷, and Arg⁴⁴⁸. Only residues A5–A6 in the DNA template are shown. Dashed lines indicate hydrogen bonds. (f) The insertion frameshift error observed in simulation XIII (R448A, purple) compared to wild-type simulation I (red). The frameshift behavior can be described in three steps: 1), A5 and A6 rotate/shift toward the downstream side; 2), Gln⁴⁴⁰ flips to A5; and 3), dTTP shifts toward the upstream side and flips to A6. Nucleotides are drawn with a smaller bond radius. Dashed lines indicate hydrogen bonds.

Figure 3

Active-site and dTTP-binding pocket coordination and rearrangement in pol μ. (a) Representative active-site conformation of pol μ from simulation I. Bold dashed lines indicate coordination around Mg²⁺; thin dashed lines indicate hydrogen bonds between water molecules and the DNA/dTTP. Mg²⁺(A), catalytic ion; Mg²⁺(B), nucleotide-binding ion. (b) Comparison of His³²⁹-Asp³³⁰ in pol μ in simulations I (red, with dTTP) and II (green, without dTTP). Bold dashed lines indicate coordination around Mg²⁺, black thin dashed lines indicate hydrogen bonds formed in simulation I, and blue thin dashed lines indicate hydrogen bonds formed in simulation II. (c) The location of the dTTP-binding pocket in pol μ (red, Gln⁴⁴⁰ and Glu⁴⁴³ in simulation I; green, Gln⁴⁴⁰ and Glu⁴⁴³ in simulation II; blue, A5 and T17 in DNA). Dashed lines indicate the distances used in cluster analysis (Fig. 3f). (d) Comparison of the dTTP-binding pocket of pol μ (left) and pol λ (right). Structures with dTTP are marked red (simulation I of pol μ, and ternary crystal structure of pol λ); structures without dTTP are marked green (simulation II of pol μ, and binary crystal structure of pol λ). Dashed lines indicate hydrogen bonds. The names of residues controlling the accommodating process are shown in red. (e) Comparison of active-site conformation of pol μ in simulations I (red and blue, with dTTP) and II (green, without dTTP). Asp³³⁰ in simulation I is marked blue. (f) Cluster analysis of Gln⁴⁴⁰ (left) and Glu⁴⁴³ (right) in simulations I (red) and II (black) based on dihedral angles (CA-CB-CG-CD) and distances to the DNA (Gln⁴⁴⁰:NE2-T17:C5M or Glu⁴⁴³:CD-A5:C2).