Distal Mutations in the Beta-clamp of DNA Polymerase III* Disrupt DNA Orientation and Affect Exonuclease Activity

04 November 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

DNA polymerases are responsible for the replication and repair of DNA found in all DNA-based organisms. DNA Polymerase III is the main replicative polymerase of E coli and is composed of over 10 proteins. A subset of these proteins (Pol III*) includes the polymerase (alpha), exonuclease (epsilon), clamp (beta) and accessory protein (theta). Mutations of residues in, or around the active site of the catalytic subunits (alpha and epsilon) can have significant impact on catalysis. However, the effects of distal mutations in non-catalytic subunits on the activity of catalytic subunits are less well characterized. Here, we investigate the effects of two Pol III* variants: beta-L82E/L82'E and beta-L82D/L82'D, on the proofreading reaction catalyzed by epsilon. MD simulations reveal major changes in the dynamics of Pol III*, that extend throughout the complex. These changes are mostly induced by a shift in the position of the DNA substrate inside the beta-clamp, although no major structural changes are observed in the protein complex. QM/MM calculations indicate that the beta-L82E/L82'D variant has reduced catalytic proficiency due to highly endoergic reaction energies resulting from structural changes in the active site and differences in the electric field at the active site arising from the protein and substrate. Conversely, the beta-L82E/L82'E variant is predicted to maintain proofreading activity, exhibiting a similar reaction barrier for nucleotide excision compared with the WT system. However, significant differences in the reaction mechanism are obtained due to the changes induced by the mutations on the beta-clamp.

Keywords

MD
QM/MM
polymerase

Supplementary materials

Title
Description
Actions
Title
Analyses of Both Short and Long DNA Systems Studied
Description
Additional analyses for all systems studied including RMSD, RMSF, matrix correlation, normal modes, EDA, NCI, ELF, ESP as well as network analysis data.
Actions
Title
Animations of Normal Modes and Reaction Paths
Description
Three videos of the first normal mode for all three systems studied, the reaction path for the WT and the reaction path for the beta-L82E/L82'E system.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.