Large tumor antigen (T antigen) was extracted from SV40‐infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono‐ or dimeric 4‐6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen‐specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen‐specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.

It has been proposed that p68, a nuclear protein of relative molecular mass 68,000, functions in the regulation of cell growth and division. A complementary DNA analysis of the protein has revealed extensive amino-acid sequence homology to the products of a set of genes recently identified in organisms as diverse as Escherichia coli and man, which include the eukaryotic translation initiation factor elF-4A. The protein products of the new gene family have several motifs in common which are thought to be involved in nucleic acid unwinding. As yet, however, only elF-4A, through its effect on RNA, has been shown to possess unwinding activity. Here we report that purified p68 also exhibits RNA-dependent ATPase activity and functions as an RNA helicase in vitro. The protein was first identified by its specific immunological cross reaction with the simian virus 40 large T antigen, the transforming protein of a small DNA tumour virus. Surprisingly, T antigen also has an RNA-unwinding activity: the homology between the two polypeptides, although confined to only a small region resembling the epitope of the cross-reacting antibody (PAb204), should therefore be of functional significance. Furthermore, the RNA-unwinding activity may be involved in the growth-regulating functions of both proteins.

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'Corresponding author Communicated by R.Knippers p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti-helicase activity can be explained by our finding that p53 binds with high affinity to singlestranded nucleic acids and has a strong DNA-DNA and RNA-RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA-RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to singlestranded DNA, imply an RNA-specific function of p53 in vivo.

The DEAD box proteins encoded by the genes ddx5 (p68) and ddx17 (isoforms p72 and p82) are more closely related to each other than to any other member of their family. We found that p68 negatively controls p72/p82 gene expression but not vice versa. Knocking down of either gene does not affect cell proliferation, in case of p68 suppression, however, only on condition that p72/p82 overexpression was granted. In contrast, co-silencing of both genes causes perturbation of nucleolar structure and cell death. In mutant studies, the apparently redundant role(s) of p68 and p72/p82 correspond to their ability to catalyze RNA rearrangement rather than RNA unwinding reactions. In search for possible physiological targets of this RNA rearrangement activity it is shown that the nucleolytic cleavage of 32S pre-rRNA is reduced after p68 subfamily knock-down, most probably due to a failure in the structural rearrangement process within the pre-60S ribosomal subunit preceding the processing of 32S pre-rRNA.