Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Abstract

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.

Maintenance of genomic integrity by p53: complementary roles for activated and non-activated p53

“…Therefore, it is possible that the p53 population not engaged in transcriptional regulation could exert functions other than induction of growth arrest or apoptosis, and directly participate in processes of DNA repair via its various biochemical activities described above. The exonuclease activity, for example, could be involved in repair processes such as DSB repair, which is thought to require DNA helicases, like the Ku autoantigen (Suwa et al, 1994;Tuteja et al, 1994), and exonucleases. Furthermore, p53 might act as an external proofreader for errors produced by cellular DNA polymerases involved in DNA replication and DNA repair also under DNA damage conditions.…”

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein

“…Polyclonal antibodies directed against Ku86 and Ku70 (designated Ku86 pc and Ku70 pc in our study) were provided by Dr Falaschi (UNIDO, Trieste, Italy) (Tuteja et al, 1994); Human serum (AF) that contained autoantibodies that cross-reacted with Ku70 and Ku86 antigens (Francoeur et al, 1986) were provided by Dr EM Tan (the Scripps Research Institute, La Jolla, USA) and puri®ed antibodies were obtained in our laboratory by the protein A sepharose procedure.…”

“…Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini or other discontinuities in the DNA structure without apparent sequence speci®city (Minori and Hardin, 1986;Blier et al, 1993;Falzon et al, 1993). Ku has also been shown to possess DNA-dependent ATPase (Cao et al, 1994) and helicase (Tuteja et al, 1994) activities. Lastly, Ku has also been reported to be capable of sequence-speci®c binding within the promoter elements of genes (Gri n et al, 1996).…”

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

“…22 Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini in the DNA structure without apparent sequence specificity. [23][24][25] Ku has also been shown to possess DNA-dependent adenosine triphosphatase 26 and helicase 27 activities. In vitro, heterodimerization of active Ku requires the cotranslation of both subunits.…”