Effect of mutant small, acid-soluble spore proteins containing cysteine or tryptophan on DNA properties in vivo and in vitro.
Two derivatives of the alpha/beta-type small acid-soluble spore protein (SASP) SspCwt have been constructed, each containing a residue potentially useful for physico-chemical analysis of protein-protein or protein-DNA interactions. In one mutant protein (SspCtrp) residue 27 (Met) was replaced by Trp; in the second (SspCcys) residue 48 (Asn) was replaced by Cys. Both mutant proteins were expressed in Bacillus subtilis spores at levels similar to those of SspCwt, and SspCcys and SspCtrp restored ultraviolet light (UV) resistance and plasmid negative supercoiling in spores lacking major alpha/beta-type SASP to levels similar to those restored by SspCwt. While the purified mutant proteins bound more weakly to DNA than SspCwt, all three had the same relative affinity for different DNAs, ie poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than pUC19, and purified SspCcys and SspCtrp gave the same pattern of DNase protected bands with pUC19 as SspCwt. Binding of SspCcys or SspCtrp to poly(dG).poly(dC) in vitro also prevented the formation of cyclobutane type cytosine dimers upon UV irradiation, as does binding of SspCwt. These data indicate that the two mutant proteins are extremely similar to SspCwt in their interaction with DNA, and thus may be useful in probing SASP-SASP and SASP-DNA interactions directly by physical or chemical techniques. Indeed, binding of SspCtrp to poly(dG).poly(dC) resulted in a 2.5-fold enhancement of the proteins Trp fluorescence.
Sanchez-Salas
J L
JL
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Sharon
M
M
Setlow
P
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011740:Pyrimidine Dimers; D012808:Sigma Factor; D013050:Spectrometry, Fluorescence; D013171:Spores, Bacterial / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011089:Polydeoxyribonucleotides; D011740:Pyrimidine Dimers; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D014364:Tryptophan; D003545:Cysteine