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Vol. 10, Issue 8, 2655-2668, August 1999
*Laboratory of Molecular Genetics, National Institute on Aging,
National Institutes of Health, Baltimore, Maryland 21224; and
Werner syndrome (WS) is a human progeroid syndrome characterized by
the early onset of a large number of clinical features associated with
the normal aging process. The complex molecular and cellular phenotypes
of WS involve characteristic features of genomic instability and
accelerated replicative senescence. The gene involved
(WRN) was recently cloned, and its gene product (WRNp)
was biochemically characterized as a helicase. Helicases play important
roles in a variety of DNA transactions, including DNA replication,
transcription, repair, and recombination. We have assessed the role of
the WRN gene in transcription by analyzing the
efficiency of basal transcription in WS lymphoblastoid cell lines that
carry homozygous WRN mutations. Transcription was
measured in permeabilized cells by [3H]UTP incorporation
and in vitro by using a plasmid template containing the RNA polymerase
II (RNA pol II)-dependent adenovirus major late promoter. With both of
these approaches, we find that the transcription efficiency in
different WS cell lines is reduced to 40-60% of the transcription in
cells from normal individuals. This defect can be complemented by the
addition of normal cell extracts to the chromatin of WS cells. Addition
of purified wild-type WRNp but not mutated WRNp to the in vitro
transcription assay markedly stimulates RNA pol II-dependent
transcription carried out by nuclear extracts. A nonhelicase domain (a
direct repeat of 27 amino acids) also appears to have a role in
transcription enhancement, as revealed by a yeast hybrid-protein
reporter assay. This is further supported by the lack of stimulation of
transcription when mutant WRNp lacking this domain was added to the in
vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS
cells may be a primary molecular defect responsible for the WS clinical phenotype.
Pathology Department, University of Washington School of
Medicine, Seattle, Washington 98195-7470
These authors contributed equally to this work.
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