Identification of a Tus protein segment that photo-cross-links with TerB DNA and elucidation of the role of certain thymine methyl groups in the Tus-TerB complex using halogenated uracil analogues.
Six potential hydrophobic sites of the Tus-TerB complex were analyzed using bromodeoxy-uridine and iododeoxyuridine as isosteric analogues of thymine. Analogues were incorporated at individual sites, and dissociation rates were measured in 150 mM potassium glutamate, pH 8.0, using a nitrocellulose filter assay. These halogenated analogues serve as a probe of the environment in which they reside. Our measurement revealed at least two types of responses. Three sites showed increases in stability with the introduction of the bromo and iodo derivatives. The enhanced stability is proposed to result from polar or charged molecules in the vicinity of the halogenated analogues through dipole-dipole, dipole-ion, or dipole-induced dipole interactions. The other three sites exhibited the opposite trend, being destabilized by the introduction of these analogues. The destabilizing effects are attributed to a hydrophobic environment which cannot accommodate polar molecules. The photoreactivity of these analogues was utilized to specifically cross-link the Tus protein and TerB DNA. Using the substitution of bromodeoxyuridine at position 8 in the TerB DNA, Tus protein was covalently attached to the DNA, and by trypsin digestion a fragment of Tus was isolated. Sequencing of the peptide fragment revealed a segment that matched the amino acid composition from 122-139 of the Tus protein.
Duggan
L J
LJ
Department of Bioscience and Biotechnology, Drexel University, Philadelphia Pennsylvania 19104, USA.
Asmann
P T
PT
Hill
T M
TM
Gottlieb
P A
PA
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001973:Bromodeoxyuridine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005971:Glutamates / Q000378:metabolism; D006207:Half-Life; D007065:Idoxuridine / Q000378:metabolism; D013941:Thymine; D014498:Uracil / Q000031:analogs & derivatives*
D001426:Bacterial Proteins; C062011:DNA replication terminus site-binding protein, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005971:Glutamates; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D014498:Uracil; D004247:DNA; D001973:Bromodeoxyuridine; D007065:Idoxuridine; D013941:Thymine