Guzder S N, Sung P, Prakash L, Prakash S
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
J Biol Chem. 1999 Aug 20;274(34):24257-62. doi: 10.1074/jbc.274.34.24257.
Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.
酿酒酵母RAD4、RAD7、RAD16和RAD23基因在紫外线（UV）损伤DNA的核苷酸切除修复（NER）中发挥作用。先前的生化研究表明，Rad4和Rad23蛋白以化学计量复合物的形式结合，该复合物名为NEF2，而Rad7和Rad16蛋白形成另一种化学计量复合物，名为NEF4。虽然在体外重组系统中，NEF2对于切割UV损伤的DNA是必不可少的，但NEF4会刺激切割反应。NEF2和NEF4都能结合UV损伤的DNA，这引发了一种有趣的可能性，即这两种复合物协同作用，以实现体内核苷酸切除修复所需的对DNA损伤界定的高度特异性。与这一假设一致，我们发现NEF2和NEF4以协同方式结合UV损伤的DNA，该反应依赖于ATP。我们还纯化了Rad7蛋白，并表明它能结合DNA，但对UV损伤的DNA没有偏好。Rad7与NEF2发生物理相互作用，表明Rad7在将NEF2与NEF4连接起来方面发挥作用。
