A MUTATION IN ESCHERICHIA-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 sin gle-stranded (ss) DNA and functions in replication, recombination and repair. Curth et al, (Biochemistry, 32 (1993) 2585-2591) have shown th at a mutant SSB protein, in which Trp-54 has been replaced by Ser (W54 S) in each subunit, binds preferentially to ss-polynucleotides in the (SSB)(35) mode in which only 35 nucleotides are occluded per tetramer under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) mo de. The W54S mutant also displays increased UV sensitivity and slow gr owth phenotypes, suggesting defects in vivo in both repair and replica tion (Carlini et al. (Molecular Microbiology, 10 (1993) 1067)). We hav e 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 prefe rence. 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 (SS B)(35) mode (omega(35)) are altered significantly compared to wtSSB. T he increased intra-tetramer negative cooperativity makes it more diffi cult for ss-DNA to bind the third and fourth subunits of the W54S tetr amer, explaining the increased stability of the (SSB)(35) mode in comp lexes with poly(dT). When bound to dA(pA)(69) in the (SSB)(35) mode, W 54S tetramer also displays a dramatically lower inter-tetramer positiv e cooperativity (omega(35) = 77(+/- 20)) than wtSSB (omega(35) greater than or equal to 10(5)) as well as a significantly lower affinity for ss-DNA. These results indicate that a single amino acid change can dr amatically influence the ability of SSB tetramers to bind in the diffe rent SSB binding modes. The altered ss-DNA properties of the W54S SSB mutant are probably responsible for the observed defects in replicatio n and repair and support the proposal that the different SSB binding m odes may function selectively in replication, recombination and/or rep air.