Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase.
Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster [J.J.Sekelsky, M.H.Brodsky, G.M. Rubin and R.S. Hawley (1999) Nucleic Acids Res., 27, 3762-3769]. The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms. Two of these isoforms are almost identical and have a predicted molecular weight of 54 kDa. The third isoform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region. A small isoform of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified. The purified protein is a single-stranded DNA-stimulated ATPase (dATPase) and a 3'-->5' DNA helicase. Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwinding reaction better than other NTPs. The turnover number for the single-stranded DNA-stimulated dATPase activity was 1380 min(-1), approximately 1.5-fold higher than that observed for the ATPase activity (900 min(-1)). The purified protein catalyzed unwinding of partial duplex substrates up to at least 93 bp, however, unwinding of an 89 bp blunt duplex substrate was not detected.
Ozsoy
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AZ
Curriculum in Genetics and Molecular Biology, Department of Biology and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.
Sekelsky
J J
JJ
Matson
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SW
D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D043583:Nucleoside-Triphosphatase; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D053484:RecQ Helicases; D013379:Substrate Specificity
D007527:Isoenzymes; D009841:Oligonucleotides; C506732:RECQL5 protein, human; D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D053484:RecQ Helicases