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.

Sustained active site rigidity during synthesis by human DNA polymerase μ

“…Subtle adjustments in the position of the side chains of Val420 and Trp434, as well as His329 and Asp330 correlate with incoming nucleotide binding and metal site occupation. The movement of the two latter side chains is consistent with previous molecular dynamics simulations of the mPol μ ternary complex structure 39 .…”

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polµ and Polλ

“…Informative crystal structures leading to this conclusion do not exist for polymerase mu (Polµ): the only one solved is a ternary complex with gapped DNA and incoming nucleotide [(13); PDB ID: 2IHM; Supplementary Figure S1C]. However, like for Polλ, an ab initio closed conformation of the Polµ apoenzyme is predicted by molecular dynamics simulations (14). …”

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

“…Structural and computational studies have uncovered important differences and similarities regarding how pol μ incorporate a cognate nucleotide into single-nucleotide gapped DNA, compared to other X-family members [29], [30]. For pol β, upon binding the cognate incoming nucleotide, the enzyme undergoes a large-scale protein motion in the thumb subdomain from open (inactive) to closed (active) conformation [31]–[33].…”

“…The large-scale protein motion in pol β and pol X or DNA motion in pol λ is crucial for the polymerization activity [31], [37], [38]. In pol μ, studies have suggested the lack of significant DNA or protein motion before chemistry [29]. Pol μ shares with pol β and pol λ the notion that subtle active-site protein residue motions help organize the conformation of the active site and prepare for the following chemical step [39], but the specific residues are different [29], [33], [36].…”