A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells.
In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.
Hughes
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CNRS-IFC1, Institut de Recherches sur le Cancer, UPR 9044, 7 rue Guy Moquet, BP 8, 94801 Villejuif Cedex, France. hughes@infobiogen.fr
Baldacci
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D051716:Replication Protein A; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D000251:Adenosine Triphosphatases; D004265:DNA Helicases