Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme act ivities of the Sts1 mutant were temperature-sensitive in vitro and were red uced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, whic h was temperature-sensitive. In contrast the Sts2 mutant did not show diffe rences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 s ubunit, the HsdSts2 subunit was not able to compete with the wild-type subu nit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutatio ns map two important regions in HsdS subunit responsible for DNA-protein an d protein-protein interactions, respectively. (C) 2000 Federation of Europe an Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.