Nucleic Acids Research, Vol 27, Issue 16 3245-3252, Copyright © 1999 by Oxford University Press
JP Jost, S Schwarz, D Hess, H Angliker, FV Fuller-Pace, H Stahl, S Thiry and M Siegmann
We have shown previously that DNA demethylation by chick embryo 5-
methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino
acid sequences of nine peptides derived from a highly purified 5- MeC-DNA
glycosylase complex were identified by Nanoelectrospray ionisation mass
spectrometry to be identical to the mammalian nuclear DEAD box protein p68
RNA helicase. Antibodies directed against human p68 helicase cross-reacted
with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the
glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of
mammalian p68 was isolated and sequenced. Its derived amino acid sequence
is almost identical to the human p68 DEAD box protein up to amino acid
position 473 (from a total of 595). This sequence contains all the
essential conserved motifs from the DEAD box proteins which are the ATPase,
RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in
the C-terminal region rather diverge from the human p68 RNA helicase
sequence. The recombinant chicken DEAD box protein expressed in Escherichia
coli cross-reacts with the same p68 antibodies as the purified chicken
embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an
RNA-dependent ATPase and an ATP-dependent helicase activity. However, in
the presence or absence of RNA the recombinant protein had no 5-MeC- DNA
glycosylase activity. In situ hybridisation of 5 day-old chicken embryos
with antisense probes of the chicken DEAD box protein shows a high
abundance of its transcripts in differentiating embryonic tissues.
ARTICLES
A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5- MeC-DNA glycosylase
Friedrich Miescher-Institute, PO Box 2543, CH-4002 Basel, Switzerland. jost@fmi.ch
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