Modern Atomic Force Microscopy and Its Application to the Study of Genome Architecture

Abstract

Recent development of atomic force microscopy (AFM) has been accomplished by various technical and instrumental innovation including high-resolution imaging technology in solution, fast-scanning AFM, and general methods for cantilever modification and force measurement. These modern AFM technologies have made it possible to conduct biological studies under physiological conditions. Application of the recognition imaging mode that can simultaneously obtain a topographic image together with a recognition signal is now successful by using protein- (antibody-) coupled cantilever, and revealed the specific protein bindings on the chromatin. AFM can also be combined with biochemical and cytochemical methods. Recent AFM researches involving series of reconstitution experiments have shown that the efficiency of the chromatin reconstitution by salt-dialysis method is drastically increased simply by using longer ( > 100 kb) and supercoiled DNA. This suggests that the physical properties of DNA are critical for the higher-order chromatin folding. Since double-stranded DNA, like other polymer chains, carries certain elasticity and flexibility, the length of DNA could affect the stability of nucleosome and chromatin fiber. This notion is well supported by the fact that in eukaryotic chromosome, the averaged length of a single chromatin loop is ∼ 100 kb. On the contrary, the large-scale structure of chromatin fiber is affected by a local protein binding. Indeed, histone H1 is essential for the reconstitution of 30 nm fibers. Type II topoisomerase (Topo II) has been known as a major component of chromosomal scaffold and an essential protein for mitotic chromosome condensation. AFM has shown that Topo II binds to bear DNA and clamps two DNA strands even in the absence of ATP, and promotes chromatin compaction depending on the existence of histone H1. Namely, H1-induced 30-nm chromatin fibers were converted into large complex by the effect of Topo II. On the basis of these results, a chromatin packing model triggered by Topo II that clamps DNA strands can be proposed. AFM analyses have also shown the similarities and differences between the eukaryotic and prokaryotic genome organizations. In spite of the presence of different structural proteins, the higher-order stepwise hierarchies from 30 nm fiber to 80 nm beaded structure are shared in eukarya, bacteria, and archaea, but the fundamental structural units are very different, that is, nucleosomes in eukarya and some archaea, and nonnucleosome unit in bacteria.