Nucleic Acids Research, Vol 25, Issue 19 3881-3888, Copyright © 1997 by Oxford University Press
P Hughes and G Baldacci
In an effort to identify cellular helicases that mimic the action of SV40
large T-antigen, we performed replication protein A (RPA) affinity
chromatography on cell extracts from the mouse mammary carcinoma cell line
FM3A. In this way, a novel DNA helicase was isolated and purified to near
homogeneity. The most purified fractions showed the presence of two
proteins of 28 and 21 kDa. Both proteins interacted with 32P- labeled
partially duplex DNA when bound to nitrocellulose membranes and were
efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was
strongly stimulated by RPA on DNA substrates containing duplex regions
longer than 18 bp. Only weak stimulation was observed in the presence of
Escherichia coli single strand DNA binding protein (SSB). The enzyme
unwinds DNA in the 5'-3' direction in relation to the strand to which it
binds. Only ATP and dATP were efficient as nucleoside triphosphate
co-factors, and showed similar Km values of approximately 0.6 mM. The
properties of this enzyme suggest that it may take part in reactions
mediated by RPA such as those predicted to occur at replication forks or
alternatively may function during DNA repair or recombination.
ARTICLES
A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells
CNRS-IFC1, Institut de Recherches sur le Cancer, UPR 9044, 7 rue Guy Moquet, BP 8, 94801 Villejuif Cedex, France. hughes@infobiogen.fr
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