Sandigursky M, Mendez F, Bases R E, Matsumoto T, Franklin W A
Department of Radiology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Radiat Res. 1996 May;145(5):619-23.
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.
先前已证明，脱氧核糖磷酸二酯酶（dRpase）活性与DNA修复酶核酸外切酶I相关，并且通过添加大肠杆菌单链DNA结合蛋白（Ssb）可刺激该活性。这种活性催化DNA中无嘌呤/无嘧啶（AP）位点处的脱氧核糖磷酸基团的释放，这些位点已通过AP内切核酸酶的作用被清理。我们现在使用酵母双杂交系统来证明核酸外切酶I和Ssb之间发生了蛋白质-蛋白质相互作用。当大肠杆菌ssb基因与GAL4转录激活因子的DNA激活结构域框内融合，且核酸外切酶I基因与DNA结合结构域框内融合时，通过酵母在选择培养基上的生长以及β-半乳糖苷酶活性的测定，确定产生了功能性GAL4转录激活因子。我们还使用在切口AP位点处含有多个链中断的双链噬菌体M13 DNA证明了Ssb可以刺激核酸外切酶I的dRpase活性。这些结果表明，Ssb可能是细菌中有效碱基切除修复所必需的。
