An intramolecular DNA triplex is disrupted by point mutations associated with hereditary persistence of fetal hemoglobin.

T.J. Ley,M.J. Ulrich,W.J. Gray

doi:10.1016/s0021-9258(19)37010-3

Abstract

Several investigators have suggested that secondary structures in DNA may be involved with physiologic gene regulatory processes in higher organisms. This hypothesis has been difficult to prove, however, since naturally occurring mutations that alter secondary DNA structures have not yet been identified. In this report, we describe a secondary DNA structure upstream from the human gamma-globin genes; this structure is formed in a homopyrimidine-homopurine tract and is stabilized by acidic pH and negative supercoiling of plasmid DNA. Since this structure is asymmetrically cleaved by S1 nuclease, it probably contains a single-stranded region and an intramolecular triplex. The single-stranded region is actually accessible for Watson-Crick base pairing with exogenous oligomers, a characteristic that permitted us to directly map the secondary DNA structure without additional chemical modifications of the supercoiled DNA. Five different point mutations just downstream from the single-stranded region are associated with hereditary persistence of fetal hemoglobin; four of these mutations dramatically reduce the stability of the secondary DNA structure, suggesting that these mutations alter formation of the intramolecular triplex by destabilizing critical Hoogsteen (triple-stranded) base pairs. These mutations may therefore represent a novel class of genetic defects that alter gene expression by changing the interaction of a critical regulatory molecule with a secondary DNA structure.

Highlights

We identify a secondary DNA structure upstream from the human y-globin genes that is stabilized by negative supercoiling and acidic pH
Our observations suggest that these conditions stabilize an intramolecular triplex and that a single-stranded region just upstream from this triplex
HPFH MutaAtilotners a Secondary DNA Structure can bind to exogenously added single-stranded DNA via Watson-Crick base pairing. This secondary DNA structure is disrupted by four separate point mutations that are associated with the syndrome of hereditary persistence of fetal hemoglobin

Summary

Present address

Escherichia coli was incubated with 30 units of topoisomerase I (calf thymus, BRL) in a 100-p1 reaction containing 50 mM Tris, pH 7.5, 50 mM KC1, 10 mM MgC12,0.1 mM EDTA, 0.5 mM dithiothreitol, and 0-120 pg/ml ethidium bromide) at 37 “C for 1 h; the plasmid DNA was extracted withphenol/chloroform One pg of this material (-1 PM) was incubated for 30-60 min a t 37 “C in a 15-pl reaction site in supercoiled plasmids (Gray andLey, 1985);this obser- containing 100 mM NaOAc, pH 4.5, with 20,000 cpm of an end-labeled vation was made independently by Tate et al (1986), who suggested that this site was cleaved symmetrically on both strands of DNA.

MATERIALS AND METHODS

RESULTS

A Model for the Secondary DNA Structure of the -200

DISCUSSION