Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.
A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.
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Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Lee
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Hurwitz
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017886:Durapatite; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D017886:Durapatite; D000251:Adenosine Triphosphatases; D004265:DNA Helicases