DNA Repair 2 (2003) 187–198
Identification of the human HEX1/hExo1 gene promoter and
characterization of elements responsible for promoter activity
Paula D. Ladd
a
, David M. Wilson, III
c
, Mark R. Kelley
a,b
, David G. Skalnik
a,b,∗
a
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
b
Section of Pediatric Hematology/Oncology, Department of Pediatrics, Herman B Wells Center for Pediatric Research,
Indiana University School of Medicine, Indianapolis, IN 46202, USA
c
Laboratory of Molecular Gerontology, GRC, National Institute on Aging, NIH, 5600 Nathan Shock Drive,
Baltimore, MD 21224-6825, USA
Accepted 4 October 2002
Abstract
HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5
to 3
exonuclease and flap structure-specific endonuclease
activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone
marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter
fragment extending from −6240 to +1600 bp exhibits cell-type specific activity in transient transfection assays. This fragment
directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but
low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified
a fragment spanning −688 to +1600 bp that exhibits full transcriptional activity while a slightly shorter fragment from −658
to +1600 bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that
interact with −687 to −681 bp and −665 to −658 bp elements. Oligonucleotide competition and antibody disruption studies
determined that the transcription factor CREB-1 recognizes the −687 to −681 bp element, while transcription factors Sp1 and
Sp3 recognize the −665 to −658 bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces
HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: HEX1/hExo1; Electrophoretic mobility shift assay; Transient transfection; Hematopoiesis; DNA recombination and repair; RAD2
nucleases
1. Introduction
Chromosome processing is central to several
critical biological events, such as DNA repair, recom-
bination and replication. In DNA repair, enzymes op-
erate to excise inappropriate or damaged nucleotides
∗
Corresponding author. Tel.: +1-317-274-8977;
fax: +1-317-274-8679.
E-mail address: dskalnik@iupui.edu (D.G. Skalnik).
or to incise at modified sites or incorrect base pairs
[1]. Such events promote the immediate or eventual
removal of the target damage, preventing replication
errors and the fixation of genetic mutations. During
recombination, nucleolytic enzymes act to generate
single-stranded DNA regions for strand exchange or
direct annealing and ligation, or to process ‘flap-like’
(bifurcated) DNA structures formed as recombination
intermediates [2–5]. Recombination is critical for
both repairing potentially hazardous genetic damage
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