Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA.

Amna Abdalla Mohammed Khalid,Silvia Onesti,Barbara Medagli,Loredana Casalis,Pietro Parisse

doi:10.3390/ma14030687

Abstract

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

Highlights

Understanding at the molecular level the mechanisms that govern DNA replication in proliferating cells is fundamental to understand diseases connected to genomic instabilities, such as genetic syndromes and cancer
In order to address conformational changes of double-stranded DNA upon interaction with the MCM protein complex to distinguish between different binding modes, we initially characterized the conformation of DNA alone on the mica surface by analyzing the end-to-end distance/contour length correlation, as described in Materials and Methods
Representative atomic force microscopy (AFM) images taken in air conditions are shown for three different DNA

Summary

Introduction

Understanding at the molecular level the mechanisms that govern DNA replication in proliferating cells is fundamental to understand diseases connected to genomic instabilities, such as genetic syndromes and cancer. Atomic force microscopy (AFM) has proven very useful to visualize and characterize the interaction between proteins and nucleic acids in the physiological environment, through the careful statistical analysis of single DNA molecules contour length and bend angle variations upon interactions [1,2,3,4,5,6]. Materials 2021, 14, 687 those, atomic force microscopy (AFM) has proven very useful to visualize and characterize the interaction between proteins and nucleic acids in the physiological environment, through the careful statistical analysis of single DNA molecules contour length and bend angle variations upon interactions [1,2,3,4,5,6]

Methods

Results

Conclusion