Nucleic Acids Research, Vol. 20, No. I
A CT promoter element binding protein: definition of a
double-strand and a novel single-strand DNA binding motif
Rukmini Kolluri, Ted Albert Torrey and Alan J.Kinniburgh*
Department of Human Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo,
NY 14263, USA
Received September 10, 1991; Revised and Accepted November 22, 1991
Numerous genes contain promoter elements that are
nuclease hypersensitive. These elements frequently
possess polypurine/polypyrimidine stretches and are
usually associated with altered chromatin structure. We
have previously isolated a clone that binds a class of
CT-rich promoter elements. We have further
characterized this clone, termed the nuclease-sensitive
element protein-1, or NSEP-1. NSEP-1 binds both
duplex CT elements and the CT-rich strand of these
elements in a 'generic' sequence specific manner and
has overlapping but distinct single-and double-strand
DNA binding domains. The minimal peptide region
sufficient for both duplex and single-strand DNA
binding includes two regions rich in basic amino acids
flanking an RNP-CS-1 like octapeptide motif. Deletion
analysis shows that the single-strand DNA binding
activity is mediated by the RNP-CS-1 like octapeptide
motif and is the key peptide region necessary for
single-strand binding. NSEP-1's affinity for CT rich
promoter elements with strand asymmetry in addition
to its double- and single-strand DNA binding properties
suggests that it may be a member of a class of DNA
binding proteins that modulate gene expression by
their ability to recognize DNA with unusual secondary
Numerous genes contain cis-acting promoter elements that are
nuclease hypersensitive. These regions are nucleosome free and
are therefore hypersensitive to DNase I (1-5). Many of these
elements are also sensitive to single-strand-specific nucleases.
These dually sensitive elements have a strong purine/pyrimidine
strand asymmetry and have been termed CT elements. The basis
of these elements' nuclease sensitivities has been ascribed to an
unusual DNA conformer. This conformer, termed the H-DNA
form, is an intramolecular triplex/single-stranded structure. One
of us (6) has previously shown that a CT promoter element of
the c-myc gene can assume the H-DNA conformation under
certain conditions in vitro. These conditions include low pH
and/or high levels of supercoiling which make it unlikely that
H-DNA forms spontaneously in vitro. There is also an alternative
intramolecular, triplex that may form with a G0G *C structure
(1,lA). We have therefore worked under the hypothesis that
cellular factors may induce these changes and produce a nuclease-
sensitive conformer (H-DNA or G *G *C triplex) in vivo. We have
previously isolated a clone that binds to a related set of CT
promoter elements (7). Here we define the binding specificity
of this factor, termed nuclease-sensitive element protein-I or
NSEP-1. We show that NSEP-1 binds both duplex CT elements
and the CT-rich single strand of these elements in a 'generic'
sequence-specific manner. That these are separable binding
activities is demonstrated by the creation of mutations that disrupt
single-strand binding but do not disrupt duplex CT element
MATERIALS AND METHODS
Expression of NSEP-1 proteins and deletion derivatives
A full length cDNA clone was constructed in the pET expression
vectors system (8,9) from sub-clones of the EcoRI fragments of
the NSEP-1 phage clone in pUC13. Expression of this construct
should produce a fusion protein containing 14 amino acids derived
from the phage T7 gene 10 product of the expression vector, 6
amino acids from the pUC13 multiple cloning site lacZ coding
sequence and 22 amino acids derived from the NSEP-1 5'
untranslated region which is in frame with the methionine initiator
codon (amino acid 1) of the 322 amino acid NSEP-1 protein.
Deletion derivatives of this NSEP-1 expression fusion protein were
engineered by restriction fragment deletion where convenient, or
by polymerase chain reaction employing as primers synthetic
oligonucleotides complementary to specific portions of the NSEP-1
recognition sites for sub-cloning into the expression vectors.
Expression of the recombinant proteins in E. coli was performed
as per Studier et al. (9). All deletion constructs refer to the amino
acids ofthe mature proteins. All amino terminaly deleted NSEP-1
derivatives lack the 22 amino acids ofthe 5' untranslated NSEP-l
region but retain the d10 gene and linker amino acids. Production
of extracts of the recombinant protein-producing bacteria wasby
the procedure of Landschulz et al. (10).
*To whom correspondence should be addressed
k.. 1992Oxford UniversityPress
116 Nucleic Acids Research, Vol. 20, No. I
purine-rich strand ofthe Watson-Crick base-pairs can Hoogsteen
base pair with a portion ofthe pyrimidine-rich strand thus forming
a triplex. The triplex forms in vitro in supercoiled DNA at low
pH. The dependency on low pH is due to the need for a proton
in the formation of the C+G Hoogsteen base pair. This can be
overcome by high levels of supercoiling. Alternatively, these
same elements can form a G *G C triplex at neutral pH in the
presence of Mg+2. We have previously predicted that the
proteins which bind these CT rich promoter elements that have
a high degree ofpurine/pyrimidine strand asymmetry can either
induce alternate, non-B DNA structures or stabilize them. That
nuclease-sensitive elements should be considered as in vivo
structures comes from several pieces of information. 1. Some
of these elements are DNase I sensitive in isolated nuclei
indicating that these elements exclude nucleosomes. 2. DNase
I sensitivity can be eliminated by changes in gene activity. 3.
In the case of the c-myc NSE element, the factors that bind this
element disappear coincidentally with the change in nuclease
sensitivity. Taken together, we hypothesize that the factors that
bind these nuclease-sensitive, CT-rich structures help form non-B
DNA structure(s) in vivo. As stated above, the c-myc nuclease-
sensitive element's, DNase I sensitive structure is correlated with
c-myc activity and the nuclease-resistant structure is found to
correlate with c-myc down-regulation (12).
We believe that the single-strand DNA binding properties of
NSEP-1 are consistent with the c-myc nuclease-sensitive element's
ability to form a non-B DNA conformer. We envision that an
NSEP-1 molecule can bind duplex NSE and 'capture' or bind
the C-rich strand ofthe NSE as it 'breathes' or is denatured under
torsional stress. The non-B DNA conformer (whichever it may
be) may then form spontaneously or other factors may bind and
further stabilize/induce triplex formation.
Ifthe senario we discuss above occurs, then one may ask why
elements with poor H-DNA forming potential bind more
vigorously to NSEP-1 than elements that are more likely to form
H-DNA (for example the EGFR NSE has a more stable H-DNA
structure than the c-myc NSE). We believe there are several
reasons to explain these data. First, the ability to form an H-
DNA structure under conditions of low pH in vitro may have
no bearing on in vivo H-DNA formation, especially if sequence-
specific DNA binding proteins are necessary for H-DNA
formation or stability. Second, one may speculate that DNA
elements with poor H-DNA forming potential would need a
greater affinity for DNA binding proteins that would induce or
stabilize the H-DNA structure because of the poor energetics.
On the otherhand, elements that could form H-DNA more readily
might not need the stabilizing effects of a DNA binding protein
and therefore would need to bind these proteins only weakly or
not at all. In any case, we may find that there is a family ofCT
element binding proteins with overlapping affinities for these
elements, and that H-DNA formation at any one element is more
a matter of cell physiology than chemical stability.
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We thank Drs. J. Ross and W. Held for many helpful comments
during the course of this work. We also thank Dr. Simon
Williams for materials and advice on their use. We thank Donna
Rypinski for technical assistance and Nancy Frame for secretarial
assistance. This work was supported by a grant from the NIH,
CA 43661, and a Cardiovascular Discovery Award from Glaxo,