Abstract
During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme∶DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.
Highlights
DNA polymerase, which is responsible for copying DNA, is a vital enzyme involved in the transfer of genetic information for living organisms
We have performed computational simulations on DNA polymerase to understand the fundamental dynamics of the enzyme
We investigated the dynamics of DNA polymerase ‘‘opening’’ in the binary state starting from three different conformations
Summary
DNA polymerase, which is responsible for copying DNA, is a vital enzyme involved in the transfer of genetic information for living organisms. Replicative DNA polymerases can generate double-stranded DNA at rates of tens or hundreds of nucleotide additions per second while only incorrectly matching a nucleotide once every tens to hundreds of thousands of nucleotides added [1]. This level of specificity suggests the dynamics and conformations of DNA polymerase are important for proper substrate binding and catalysis. Within the Klenow fragment, the polymerase domain resembles the shape of a human hand with a thumb subdomain that grasps the DNA, a palm subdomain that contains the active site, and a mobile fingers subdomain involved in dNTP binding [3,4]. Upon binding of a dNTP (ternary state) that forms a proper WatsonCrick base pair with the template strand, the fingers domain enters
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