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J Biol Chem, Vol. 273, Issue 40, 26130-26137, October 2, 1998
,
From the The human DNA topoisomerase III
(hTOP3) gene encodes a topoisomerase homologous to the
Escherichia coli DNA topoisomerase I subfamily. To
understand the mechanisms responsible for regulating hTOP3
expression, we have cloned the 5'-flanking region of the gene coding
for the hTOP3 and analyzed its promoter activity. The presence of a
single transcription initiation site was suggested by primer extension
analysis. The hTOP3 gene promoter is moderately high in GC
content and lacks a canonical TATA box, suggesting that
hTOP3 promoter has overall similarity to promoters of a
number of housekeeping genes. Examination of the promoter sequence
indicated the presence of four Sp-1 consensus binding sequences and a
putative initiator element surrounding the transcription initiation
site. Transient expression of a luciferase reporter gene under the
control of serially deleted 5'-flanking sequences revealed that the
52-base pair region from Department of Biology and
§ Department of Biochemistry, College of Science,
Bioproducts Research Center, Yonsei University,
Seoul 120-749, Korea
326 to 275 upstream of the transcription
initiation site includes a positive cis-acting element(s) for the
efficient expression of hTOP3 gene. On the basis of gel
mobility shift and supershift assays, we demonstrated that both YY1 and
USF1 transcription factors can bind to the 52-base pair region. When
HeLa cells were transiently transfected with a mutant construct which
had disabled both YY1- and USF1-binding sites, the luciferase activity
was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3
promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as
activators in the hTOP3 promoter.
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