A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding.

E. coli SSB tetramer binds with high affinity and cooperatively to single-stranded (ss) DNA and functions in replication, recombination and repair. Curth et al. (Biochemistry, 32 (1993) 2585-2591) have shown that a mutant SSB protein, in which Trp-54 has been replaced by Ser (W54S) in each subunit, binds preferentially to ss-polynucleotides in the (SSB)35 mode in which only 35 nucleotides are occluded per tetramer under conditions in which wild-type (wt) SSB binds in its (SSB)65 mode. The W54S mutant also displays increased UV sensitivity and slow growth phenotypes, suggesting defects in vivo in both repair and replication (Carlini et al. (Molecular Microbiology, 10 (1993) 1067)). We have characterized the energetics of SSBW54S binding to poly(dT) as well as short oligodeoxyribonucleotides (dA(pA)69, dT(pT)34, dC(pC)34) to determine the basis for this dramatic change in binding mode preference. We find that the W54S mutant remains a stable tetramer; however, its affinity for ss-DNA as well as both the intra-tetramer negative cooperativity and its inter-tetramer positive cooperativity in the (SSB)35 mode (omega 35) are altered significantly compared to wtSSB. The increased intra-tetramer negative cooperativity makes it more difficult for ss-DNA to bind the third and fourth subunits of the W54S tetramer, explaining the increased stability of the (SSB)35 mode in complexes with poly(dT). When bound to dA(pA)69 in the (SSB)35 mode, W54S tetramer also displays a dramatically lower inter-tetramer positive cooperativity (omega 35 = 77(+/-20)) than wtSSB (omega 35 > or = 10(5)) as well as a significantly lower affinity for ss-DNA. These results indicate that a single amino acid change can dramatically influence the ability of SSB tetramers to bind in the different SSB binding modes. The altered ss-DNA properties of the W54S SSB mutant are probably responsible for the observed defects in replication and repair and support the proposal that the different SSB binding modes may function selectively in replication, recombination and/or repair.