Properties of two DNA helicases of human cells.

Ochem, Alexander (1999). Properties of two DNA helicases of human cells. PhD thesis The Open University.



DNA helicases are ubiquitous, non-specific dsDNA unwinding enzymes involved in all aspects of DNA metabolism. In the present work, I describe the properties of two DNA helicases of HeLa cells: those of a novel enzyme, human DNA helicase VH (HDH VII), and those of the separate subunits of human DNA helicase II.
HDH VII possesses the highest specific activity among all the helicases, so far, extracted and characterized from HeLa cells, and this activity is further stimulated nearly a hundred-fold by hRPA. However, its abundance in the cell is very low since only 1 lOpg of pure protein could be recovered from 150 grams of cultured cells. The estimated molecular weight of HDH YD in its native form is close to 90-kDa whereas SDS-PAGE analysis reveals two unequal, closely-migrating bands in the region of 32-kDa, suggesting that the protein may have a hetero-trimeric conformation. The enzyme exhibits a double polarity of translocation: a property that distinguishes it from all characterized eukaryotic DNA helicases. In spite of its high specific activity, HDH VH shows a relatively low processivity even in the presence of hRPA and cannot unwind duplexes longer than 17 base pairs.
The Ku antigen consists of two subunits of 70 and 83 kDa, separable from each other only by electrophoresis under denaturing conditions, and is endowed with both duplex DNA end-binding capacity and helicase activity. I have dissected the in vitro activities of the Ku molecule, and I report that whereas the DNA end-binding property remains a prerogative of the heterodimer, the helicase activity of the Ku molecule resides exclusively in the 70 kDa subunit, and that the helicase activity of the Ku heterodimer is stimulated upon phosphorylation by the DNA-dependent protein kinase DNA-PK.

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