The Development and Use of a DNA Polymerase Arrest Assay for the Evaluation of Parameters Affecting Intrastrand Tetraplex Formation

M Neale Weitzmann,Kerry J Woodford,Karen Usdin

doi:10.1074/jbc.271.34.20958

Abstract

We show here that a K+-dependent block to DNA synthesis is a sensitive and specific indicator of intrastrand tetraplex formation that can be used, both to identify sequences with tetraplex-forming potential and to examine parameters that affect tetraplex formation. We show that tetraplex formation is determined by a complex combination of factors including the size and base composition of its constituent loops and stems. In the process of carrying out this study we have found that the number of sequences with the ability to form tetraplexes is larger than previously thought, and that such sequences are ubiquitous in eukaryote genomes.

Highlights

Interstrand tetraplexes have been implicated in the pairing of homologous chromosomes during meiosis [4] and switch recombination in immunoglobulin genes [4], and they may play important roles in the functioning of telomeres [4, 5]
We have recently shown that a G-rich sequence from the chicken ␤-globin promoter provides a Kϩ-dependent block to DNA synthesis, whose properties are consistent with the formation of an intrastrand tetrahelical structure [15]
Since DNA synthesis in the region preceding the tetraplex-forming sequence is similar in the presence or absence of Kϩ, it is apparent that the arrest of DNA synthesis is not due to an adverse effect of Kϩ on the polymerase

Summary

Introduction

Interstrand tetraplexes have been implicated in the pairing of homologous chromosomes during meiosis [4] and switch recombination in immunoglobulin genes [4], and they may play important roles in the functioning of telomeres [4, 5]. Using a series of known tetraplex-forming model sequences, we show that the ability to block DNA synthesis in a Kϩ-dependent manner is not a peculiar property of these unusual tetraplexes, but rather is diagnostic of intrastrand tetraplexes in general This property provides a rapid and simple assay for these structures and for studying the parameters that affect tetraplex formation. This assay can be carried out under physiological conditions and can detect tetraplexes at concentrations that are orders of magnitude less than those required by classical analytical techniques such as CD-spectroscopy, x-ray crystallography, and NMR, even when tetraplexes constitute only a small fraction of DNA molecules in the population. We have used this assay to identify a number of tetraplex-forming sequences in eukaryote genomes

Methods

Results

Conclusion