Functional Properties of the Separate Subunits of Human DNA Helicase II/Ku Autoantigen

Abstract

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions.To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001).Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.The Ku autoantigen was originally isolated as a nuclear protein recognized by the sera of lupus erythematosus patients (1); it is a heterodimer made of two subunits that have been ascribed slightly different molecular masses in different labs; in this work we shall define them by the molecular masses calculated from the straight amino acid sequence, namely 83 and 70 kDa. Ku has the ability to bind specifically to the ends of duplex DNA and then slide into the duplex to form a structure similar to beads on a string (2). This molecule is essential for the recombination events necessary for the rearrangement of the immunoglobulin genes (V(D)J recombination) as well as for the repair of double strand DNA breaks caused by x-ray damage. Cell lines bearing mutations affecting the 83-kDa subunit appear to be deficient in both of these properties as well as in the duplex DNA end-binding ability of the extracted nuclear proteins (3). Accordingly, Ku83-deficient mice exhibit severe combined immunodeficiency due to T and B lymphocyte arrest at early progenitor stages (3-4). Furthermore, Ku has also been reported to be a substrate as well as a cofactor of the DNA-dependent protein kinase, which is also essential for the V(D)J recombination and x-ray repair processes (5-7).In the past, we showed that Ku is also endowed with an ATP-dependent DNA helicase activity denominated human DNA helicase II (HDH II) 1 , probably located on a different moiety of the molecule than the one involved in duplex DNA binding (8). Furthermore, after cloning of the c...

DNA-dependent PK inhibits adeno-associated virus DNA integration

“…Other functions of Ku are helicase and ATPase activities. The Ku70 subunit contains the ATPase activity and is able to perform its helicase function independently of Ku80 (18). The weak ATPase activity is stimulated by DNA-PK-mediated phosphorylation.…”

“…Although both Ku70 and Ku80 interact with DNA, Ku70 makes the more intimate interactions with its C-terminal 73-aa residues. Ku70 and Ku80 functionally depend on each other, although either subunit alone can bind DNA effectively (16)(17)(18). Interestingly, Ku has the ability to translocate along DNA molecules in an ATP-independent manner (19,20).…”

Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

“…Together, our results to date suggest that the interaction of Ku with NRE1 occurs in two steps; initial contact of Ku70, followed by a Mg 2ϩ -dependent structural transition that leads to the contact of Ku80 with DNA. Interestingly, there is a recent report (81) that indicates that Ku70 alone has helicase activity.…”

“…One intriguing possibility is that the binding of Ku to the upper strand of NRE1, or to the point of strand separation that exists in the known helicase templates, recruits a second molecule of Ku to the end of the single-stranded overhang. Ku is known to form DNA-dependent dimers and Ku helicase activity has to date been observed only under conditions where dimers can form (46,81,83,84).…”

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

“…The Ku 86 and Ku 70 proteins form a heterodimeric complex (Minori and Hardin, 1986;Francoeur et al, 1986) that binds to DNA termini or other discontinuities in the DNA helix (Francoeur et al, 1986;Falzon et al, 1993;Blier et al, 1993). Both Ku subunits are required for stable binding to DNA (Wu and Lieber, 1996;Ochem et al, 1997). Ku is also able to enter the DNA molecule in an ATP-independent manner, allowing several Ku dimers to bind to a single DNA molecule and form a multimeric complex (Zhang and Yaneva, 1992).…”