Functional dissection of DNA supercoiling factor: EF-hand domains and C-terminal HDEF motif are essential for its activity.
BACKGROUND
DNA supercoiling factor (SCF) was first identified in the silkworm as a protein capable of generating negative supercoils into a relaxed DNA in conjunction with eukaryotic topoisomerase II. Drosophila melanogaster SCF localizes to puffs on polytene chromosomes, implicating its role in gene expression. The factor is a Ca2+-binding protein with four EF-hand domains and possesses a tetrapeptide sequence HDEF at its C-terminus.


RESULTS
To clarify the roles of the domains of SCF, we carried out a functional dissection of the factor. A glutamic acid to glutamine substitution at the end of the loop in EF-hand domain II or III reduced both the Ca2+ binding and supercoiling activities; simultaneous substitutions at both sites abolished these activities. During native polyacrylamide gel electrophoresis, SCF migrated more rapidly in the presence of Ca2+ than in the presence of Mg2+ or EGTA. SCF binds directly to topoisomerase II. Deletion of the C-terminal HDEF sequence destroyed the binding and supercoiling activity.


CONCLUSIONS
Two regions of SCF play critical roles in the supercoiling activity. The C-terminal HDEF is essential for the factor binding to topoisomerase II. The EF-hand domains II and III are functional for the Ca2+ binding that induces a mobility change in the factor upon gel electrophoresis.
Kobayashi
M
M
Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan.
Hirose
S
S
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000502:physiology*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D008954:Models, Biological; D008957:Models, Genetic; D016296:Mutagenesis; D009687:Nuclear Proteins; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; C115373:DNA supercoiling factor, Drosophila; D004278:DNA, Superhelical; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D004250:DNA Topoisomerases, Type II; D002118:Calcium