Trujillo K M, Sung P
University of Texas Health Science Center and Institute of Biotechnology, San Antonio, Texas 78245, USA.
J Biol Chem. 2001 Sep 21;276(38):35458-64. doi: 10.1074/jbc.M105482200. Epub 2001 Jul 13.
Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50.Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3' to 5' exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50.Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3' and 5' single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3' single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5' strand at secondary structures formed upon DNA strand separation.
酿酒酵母的RAD50和MRE11基因是DNA双链断裂的核酸酶处理所必需的。我们在酵母细胞中过表达了Rad50和Mre11，并将它们纯化至接近均一。与遗传数据一致，我们表明纯化的Rad50和Mre11蛋白形成了稳定的复合物。在Rad50.Mre11复合物中，蛋白质成分以等摩尔量存在。Mre11具有3'至5'核酸外切酶活性，可导致单核苷酸的释放。添加Rad50不会显著改变Mre11的核酸外切功能。使用基于同聚寡核苷酸的底物，我们表明Mre11和Rad50.Mre11的核酸外切酶活性对于具有双链DNA末端的底物增强。我们已经研究了Mre11对定义的、放射性标记的发夹结构的内切核酸酶功能，这些结构也含有3'和5'单链DNA突出端。Mre11能够切割发夹和3'单链DNA尾巴。Mre11的这些内切核酸酶活性在Rad50的作用下显著增强，但仅在ATP存在的情况下。基于这些结果，我们推测Mre11核酸酶复合物可能介导在DNA链分离时形成的二级结构处5'链的核酸酶消化。
