Mechanical constraints on Hin subunit rotation imposed by the Fis/enhancer system and DNA supercoiling during site-specific recombination.
Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.
Dhar
Gautam
G
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Heiss
John K
JK
Johnson
Reid C
RC
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008957:Models, Genetic; D009154:Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D012475:Salmonella / Q000235:genetics* / Q000378:metabolism
D004278:DNA, Superhelical; D035021:Factor For Inversion Stimulation Protein; D021122:Protein Subunits; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase; D003545:Cysteine