Center for DNA Replication and Genome Instability, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Nucleic Acids Res. 2010 Nov;38(21):7611-25. doi: 10.1093/nar/gkq651. Epub 2010 Jul 25.
The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.
两种内切核酸酶 Rad27（酵母 Fen1）和 Dna2 共同参与酵母中冈崎片段的加工。Mus81-Mms4 是一种结构特异性内切核酸酶，可解决停滞的复制叉以及有毒的重组中间体。在这项研究中，我们表明 Mus81-Mms4 可以通过与 Rad27 的功能和物理相互作用来抑制 dna2 突变缺陷。Mus81-Mms4 显著刺激 Rad27 活性，这解释了它能够恢复 dna2 突变引起的生长缺陷的能力。有趣的是，Rad27 刺激 Mus81-Mms4 催化切割各种底物的速率，包括退回复制叉底物。Rad27 刺激 Mus81-Mms4 的能力不依赖于 Rad27 的催化活性，但需要 Rad27 的 C 末端 64 个氨基酸片段。这表明这种刺激是由两种蛋白质之间的特定蛋白-蛋白相互作用介导的。我们的体外数据表明，Mus81-Mms4 和 Rad27 在 DNA 复制过程中协同作用，并解决各种可能阻碍正常 DNA 复制的结构。rad27 mus81 或 rad27 mms4 双突变体协同致死的事实进一步加强了这一结论。我们讨论了 Rad27、Dna2 和 Mus81-Mms4 之间相互作用在 DNA 复制中的意义。
