Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity.
Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity.
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DOI：
10.1093/nar/gkl685
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发表时间：
2006
影响因子：
14.9
通讯作者：
Brill SJ
中科院分区：
文献类型：
--
作者：
Yang L;Mullen JR;Brill SJ
SLX 5和SLX 8编码先前基于它们在缺乏Sgs 1 DNA解旋酶的酵母细胞中的存活力的需要而鉴定的环指蛋白。已知Slx 5和Slx 8蛋白是基因组稳定性所需的，并且在酵母提取物中物理相互作用;然而，它们的生化功能是未知的。为了解决这个问题，我们纯化和表征重组Slx 5和Slx 8蛋白。在这里，我们表明，Slx 5和Slx 8形成异二聚体复合物与双链DNA（dsDNA）结合活性。单独地，只有Slx 8亚基显示这种活性。结构-功能研究表明，DNA结合活性只需要Slx 8的N-末端160个氨基酸，而不需要其C-末端环指结构域。SLX 8的等位基因，表达的环指结构域单独显示几乎完全互补酵母表明，这种DNA结合结构域是不必要的，在体内功能。与这些研究结果一致，我们表明，Slx 5免疫定位到细胞核和一部分的Slx 8蛋白共分馏与染色质。这些结果表明Slx 5-Slx 8可能直接作用于DNA以促进基因组稳定性。
SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability.
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影响因子：
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