The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation.
Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes.
Lee
C G
CG
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, NY 10021, USA.
Chang
K A
KA
Kuroda
M I
MI
Hurwitz
J
J
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004303:Dosage Compensation, Genetic; D004330:Drosophila / Q000201:enzymology / Q000235:genetics*; D029721:Drosophila Proteins; D019084:Fluorescent Antibody Technique, Indirect; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043583:Nucleoside-Triphosphatase; D011485:Protein Binding; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014960:X Chromosome / Q000235:genetics* / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C070837:mle protein, Drosophila; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D020365:RNA Helicases