Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, Gdansk, Poland.
Nucleic Acids Res. 2012 Feb;40(3):1148-59. doi: 10.1093/nar/gkr813. Epub 2011 Oct 5.
DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.
DNA 复制起始蛋白 (Reps) 会被细胞蛋白酶降解。我们研究了涉及 Rep 和蛋白酶的核蛋白复合物的形成如何影响 Rep 降解。使用了所有已知的大肠杆菌 AAA+细胞质蛋白酶和广谱质粒 RK2 的复制起始蛋白 TrfA。我们的结果表明，DNA 会影响降解过程，并且观察到的效果是相反的，并且是蛋白酶特异性的。在 ClpXP 和 ClpYQ 蛋白酶的情况下，DNA 会阻止蛋白水解，而在 ClpAP 和 Lon 蛋白酶的情况下，它会刺激该过程。ClpX 和 ClpY 不能与结合 DNA 的 TrfA 相互作用，而 ClpAP 和 Lon 的活性则通过涉及蛋白酶和 TrfA 的核蛋白复合物的形成得到增强。Lon 必须在与 DNA 接触之前与 TrfA 相互作用，或者这种相互作用可以在 TrfA 已经与 DNA 结合的情况下发生。Lon 只能在 DNA 上对 TrfA 进行降解。Lon 的缺失会导致细胞中 TrfA 的稳定性更高。
