Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase

Stefan Ilic,Sabine R Akabayov,Ron Meiry,Haribabu Arthanari,Barak Akabayov,Roy Froimovici,Dan Vilenchik,Alfredo Hernandez

doi:10.1038/s41598-017-05534-3

Stefan Ilic, Sabine R Akabayov + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-05534-3

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Abstract

Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA primase. The N-terminal primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA primase. NMR analysis revealed that upon binding Mn(II), T7 DNA primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA primase revealed a preference in the enzyme’s second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.

Highlights

In about a third of all enzymes, biological function is dependent on the presence of a divalent metal cofactor in the enzyme’s active site[1]
Using a combination of biochemical and biophysical analyses, we were able to explain the basis for elevated primer synthesis activity by Mn(II)-substituted T7 DNA primase
Our study provides insight into the essential roles played by divalent metal cofactors in enzyme activity

Summary

Introduction

In about a third of all enzymes, biological function is dependent on the presence of a divalent metal cofactor in the enzyme’s active site[1]. The formation and stability of the metal-enzyme complex depend on the properties of both the metal (valence state, ionic radius, charge-accepting ability) and the amino acids that constitute the metal binding site (net charge, dipole moment and polarizability, electron donor and acceptor ability, etc.)[3]. The effects of these properties manifest in the metal ion’s coordination number which, in conjunction with its coordination geometry (arrangement of the ligands around the metal in 3D space), determines the structure and overall properties of the enzyme-metal complexes[4]. Owing to their similarity in size and charge, the metal ions Mg(II) and Mn(II), the subjects of our study, can substitute for each other in a large variety of enzymes[8]

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