IDENTIFICATION OF AMINO-ACIDS STABILIZING THE TETRAMERIZATION OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN FROM ESCHERICHIA-COLI

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein t o tyrosine or lysine leads to cells which are UV- and temperature-sens itive. The defects of both ssbH55Y (ssb-l) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation produc ing a less stable, readily dissociating protein whose more severe repl ication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was s uppressed by spontaneous mutations that changed the glutamine at posit ion 76 or 110 to leucine, Using guanidinium chloride denaturation moni tored by sedimentation diffusion equilibrium experiments in the analyt ical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76 L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable th an wild-type SSB, Additionally the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denatu ration than wildtype SSB protein. (C) 1998 Federation of European Bioc hemical Societies.