A high-throughput assay shows that DNase-I binds actin monomers and polymers with similar affinity.
Previous conflicting reports suggest that DNase-I binds F-actin with either equal or drastically different K(D) values compared to G-actin. We developed a high-throughput DNase-I inhibition assay to determine the K(D) of DNase-I for F-actin. We confirmed that phalloidin-stabilized F-actin is protected from depolymerization by DNase-I and that the critical concentration at the pointed end of phalloidin-F-actin is 45.5+/-13.9 nM. We found that DNase-I inhibition by actin follows ultrasensitive mechanics. Using varying lengths of gelsolin-capped phalloidin-F-actin, we concluded that the affinities of DNase-I for G- and the pointed end subunits of F-actin are almost indistinguishable, such that DNase-I may not distinguish between G- and F-actin conformations.
Morrison
Scott S
SS
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Dawson
John F
JF
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D008968:Molecular Conformation; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000199:Actins; D011108:Polymers; D003850:Deoxyribonuclease I