Human DNA helicase II (HDH II) is a novel ATP‐dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3′ to 5′ direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single‐stranded DNA‐dependent ATPase activity, as well as double‐stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha‐32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross‐reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA‐dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double‐stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.

Eukaryotic chromosomes terminate in specialized structures, telomeres, that are necessary for their stability and function (for reviews, see references 7 and 61). Telomeres consist of a complex of G-rich repeated DNA and of sequence-specific DNA binding proteins (for a review, see reference 20). Formation of the complex and telomere function, therefore, have stringent DNA sequence requirements (TTAGGG in vertebrates [27,44]). Synthesis of telomeric DNA is catalyzed by telomerase, a ribonucleoprotein that utilizes a domain of its own RNA component as the template for de novo addition of nucleotides to the G-rich strand (25,26,43). This process compensates for the loss of terminal sequences occurring during semiconservative replication of linear DNA molecules (47,57) and is instrumental in keeping telomere length at an equilibrium (for a review, see reference 24).Over the last few years, experimental evidence has supported the presumed role of telomere maintenance in cell life span regulation, making telomerase an essential function for longterm or unlimited cell proliferation. Deletion of the template RNA gene, which is required for enzyme activity (26), results in progressive telomere shortening and in loss of viability of otherwise immortal yeast cells (41,52). Human somatic cells, which generally lack detectable levels of telomerase (for a review, see reference 31), undergo telomere erosion with cell division in vitro (3, 14, 28; for a review, see reference 5) and have a limited life span (29). Immortal germ line cells (33, 58) and the majority of somatic cells immortalized in vitro or in vivo, on the other hand, express telomerase and maintain their telomeres (14-16, 33, 35, 51; for reviews, see references 6 and 17). Experimental lengthening of telomeres (59) or inhibition of telomerase (21,46,53) in immortal human cells has been shown to prolong or reduce, respectively, cell proliferative capacity. Alterations in telomere structure that are independent of telomerase inhibition can also have an impact on cell growth and survival. Proteins that bind telomeric DNA are known to participate in telomere length regulation (20), and in yeast, mutation or deletion of these proteins destabilizes telomeres and impairs cell survival (36, 37). In addition, in both Tetrahymena thermophila and yeast, mutations of the template domain of the telomerase RNA and the consequent expression of a mutant enzyme cause loss of telomere length regulation and of cell viability (34,41,49,60).Experimental manipulation of telomeres in lower eukaryotes has also revealed alternative mechanisms for telomere maintenance that may utilize recombination with internal telomeric repeats or gene conversion at the chromosome termini to restore telomeric sequences (38,42,56). Survivors that maintain telomeres by these processes have been consistently rescued from populations of yeast lacking a telomeric protein or telomerase, or expressing a mutant enzyme, suggesting that recombination or gene conversion may operate as a salvage pathway when the norm...

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop 11. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and 111 to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondarystructure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and 111 did. These results suggest that the sequence of the stems I and 111 impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.Keywords: catalytic RNA ; hammerhead ; trans-cleavage; RNA-binding protein.A family of highly repetitive DNA sequences, dispersed in small clusters in the newt (Notophthalmus, Triturus) genome, codes for stable, strand-specific transcripts found in both somatic and germinal tissues ([l] and Cremisi, F., personal communication). The transcripts correspond in size precisely to monomeric (330 bp) and multimeric DNA repeat units. Evidence that synthetic dimer-rize transcripts undergo site-specific, selfcatalyzed cleavage in vitro suggested that the same reaction might be involved in the processing of large multimeric transcripts in vivo 12, 31. The self-cleavage reaction requires Mg" and occurs within a conserved structure named the hammerhead, similar to that of infectious plant RNAs [4].The ovarian ribozyme transcripts differ from the analogous transcripts in other newt tissues in having an intact self-cleavage site, located about SO nucleotides from the 5' end [2]. Therefore, it seems that monomer length transcripts of the ovarian newt ribozyme ,are more likely to arise rather from trancription than from self-processing by cleavage. It has indeed been shown that the same promoter elements that regulate polymerase-11-dependent transcription of small nuclear RNAs are involved in transcription of the newt ribozyme [5, 61. The presence of an apparently functional gene that codes for the small RNA with a hammerhead domain suggests that a biological function of this particular ribozyme may be trans-cleavage of a specific, but still unknown RNA target in vivo [5, 61. Here we report an in vitro study of the trans-cleaving properties o...