Biochemical characterization of the WRN-FEN-1 functional interaction
Biochemical characterization of the WRN-FEN-1 functional interaction
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DOI：
10.1021/bi026031j
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发表时间：
2002-10-08
期刊：
影响因子：
2.9
通讯作者：
Sommers, JA
中科院分区：
文献类型：
--
作者：
Brosh, RM;Driscoll, HC;Sommers, JA
沃纳综合征是一种以染色体不稳定为特征的过早衰老疾病。最近，我们报道了WRN基因产物与人5'侧翼核酸内切酶/5'-3 '核酸外切酶（FEN-1）的新的相互作用，FEN-1是一种DNA结构特异性核酸酶，涉及对基因组稳定性重要的DNA代谢途径。为了表征WRN刺激FEN-1裂解的机制，我们确定了WRN对FEN-1裂解反应的动力学参数的影响。WRN增强了FEN-1切割的效率，而不是DNA底物结合。WRN有效地刺激FEN-1在瓣状DNA底物上的切割，其中链霉亲和素结合到上游双链体末端的末端3'核苷酸，表明WRN不需要游离的上游末端来刺激FEN-1对5'瓣状底物的切割。这些结果表明，WRN刺激FEN-1裂解的机制不同于增殖细胞核抗原和FEN-1之间的功能性相互作用。为了了解WRN-FEN-1（1）相互作用在DNA复制中的潜在重要性，我们测试了WRN对FEN-1切割冈崎片段加工过程中可能出现的几种DNA底物中间体的影响。WRN刺激FEN-1切割具有末端单核糖核苷酸、长5' ssDNA束和假Y结构的瓣底物。WRN促进FEN-1切割DNA复制/修复中间体的能力对于WRN在维持基因组稳定性中的作用可能是重要的。
Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.