Biswas E E, Zhu F X, Biswas S B
Department of Molecular Biology, University of Medicine & Dentistry of New Jersey, Stratford 08084, USA.
Biochemistry. 1997 May 20;36(20):5955-62. doi: 10.1021/bi962890u.
The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.
RTH1核酸酶参与染色体DNA的复制以及DNA损伤的修复。复制蛋白A（RPA）也是DNA复制和修复过程中不可或缺的一部分。我们研究了RPA在RTH1核酸酶功能中的作用，包括其结构特异性内切核酸酶活性。最初的体外研究使用了含有短14bp双链区域的“瓣状”或“假Y”底物，结果显示RPA的作用极小或具有抑制作用。由于对于参与DNA复制过程的蛋白质而言，抑制RPA是不合理的，我们进一步研究了这种抑制的机制。使用M13mp19单链DNA和合成寡核苷酸制备了具有长双链区域（50bp）的替代瓣状和假Y底物。酵母RPA以剂量依赖的方式刺激了RTH1核酸酶对这些底物的内切核酸酶活性。动力学分析表明，酵母RPA对核酸酶反应产生双重影响：（i）在存在RPA的情况下，RTH1核酸酶加载到DNA底物上的“加载时间”从约5分钟减少到2分钟，并且（ii）在核酸酶反应开始后，在存在酵母RPA的情况下反应的初始速率增加了10倍。对RPA与各种内切核酸酶底物相互作用的进一步分析表明，RPA具有较弱的螺旋去稳定化作用，并且可以使双链DNA的小的14bp区域解链。RTH1核酸酶在单链和双链DNA的交界处切割DNA链；因此，观察到的对小双链底物的抑制可能是由于双链解链。我们的研究还表明，RPA显著刺激了RTH1核酸酶的RNase H活性。在这两种情况下（RTH1核酸酶和RNase H），刺激可能涉及RPA与RTH1核酸酶的特异性相互作用，而不是RPA对DNA底物的结构定位。
