Protein-protein interactions between the Escherichia coli single-stranded DNA-binding protein and exonuclease I.
It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.
Sandigursky
M
M
Department of Radiology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Mendez
F
F
Bases
R E
RE
Matsumoto
T
T
Franklin
W A
WA
D017104:Bacteriophage M13; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D001616:beta-Galactosidase