Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.
The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.
Benito
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MI
Department of Biological Sciences, Stanford University, California 94305-5020, USA. benito@leland.stanford.edu
Walbot
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V
D001483:Base Sequence; D003300:Copper / Q000378:metabolism; D018983:DNA Footprinting; D019175:DNA Methylation; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D019895:Transposases; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D003300:Copper; D004254:DNA Nucleotidyltransferases; D019895:Transposases; C025205:1,10-phenanthroline