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

We have shown previously that DNA demethylation by chick embryo 5-methylcyt osine (5-MeC)-DNA glycosylase needs both protein and RNA, Amino acid sequen ces of nine peptides derived from a highly purified 5-MeC-DNA glycosylase c omplex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. An tibodies directed against human p68 helicase cross-reacted with the purifie d 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase acti vity, 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 to tal 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 div erge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 a ntibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helic ase activity, However, in the presence or absence of RNA the recombinant pr otein 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 tiss ues.