Detection of G-quadruplex DNA in mammalian cells

A Henderson,F B Johnson,Y C Huang,R M Brosh,E A Chavez,Y Wu,P M Lansdorp,D Sen,J Platt

doi:10.1093/nar/gkt957

Abstract

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

Highlights

Single-stranded guanine (G)-rich DNA can form stable secondary structures in vitro called G-quadruplex (G4) DNA [1,2]
Purified G4 samples were resuspended in phosphatebuffered saline (PBS) and monomers were resuspended in TE
Samples prepared for circular dichroism (CD) analysis were diluted in PBS or TE to a final concentration of 5–10 mM (70–95 mg/ml)

Summary

Introduction

Single-stranded guanine (G)-rich DNA can form stable secondary structures in vitro called G-quadruplex (G4) DNA [1,2]. To aid in future studies and elucidate the binding characteristics of the monoclonal antibodies for these G4 DNA structures, we deduced the amino acid sequence for the variable heavy (VH) and variable light (VL) chain of the hybridomas from the corresponding genes (Supplementary Table S2).

Results

Conclusion