DNA binding specificity and cleavage activity of Pacmmar transposase.
Mariner-like elements (MLEs) are members of the Tc1/mariner superfamily of transposable elements which transpose by a "cut and paste" mechanism. Most of the MLEs characterized to date are transpositionally inactive due to the accumulation of mutations in their transposase gene. Here, we report the biochemical study of two copies of the Pacmmar element (Pacmmar1.1 and Pacmmar1.2), isolated from the coastal crab Pachygrapsus marmoratus. These two copies present an open reading frame encoding a putative active transposase. Using an in vitro transposition assay, we show that Pacmmar transposases are unable to perform by themselves the transposition reaction. However, we demonstrate by an electrophoretic mobility shift assay that both transposases bind specifically to the inverted terminal repeat of the Pacmmar element. Moreover, an in vitro cleavage assay showed that both transposases have the capacity to cleave the transposon. The in vitro cleavage activity of Pacmmar transposases appears imprecise, suggesting the requirement of specific host factors or the presence of mutations which have modified the cleavage specificity of the enzyme.
Delaurière
Laurence
L
Université du Maine, EA2160 Mer Molécules Santé, équipe biologie moléculaire et génétique évolutive, Le Mans, F-72085 France.
Chénais
Benoît
B
Pradier
Elisabeth
E
Hardivillier
Yann
Y
Renault
Sylvaine
S
Casse
Nathalie
N
D000595:Amino Acid Sequence; D000818:Animals; D003445:Crustacea / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D013379:Substrate Specificity / Q000235:genetics; D020079:Terminal Repeat Sequences; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D004247:DNA; D019895:Transposases