Purification and properties of human DNA helicase VI.
A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
Tuteja
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N
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Ochem
A
A
Taneja
P
P
Tuteja
R
R
Skopác
D
D
Falaschi
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D002413:Cations, Divalent; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004795:Enzyme Stability; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010777:Photochemistry; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity
D000345:Affinity Labels; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D008345:Manganese; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate