Effect of flap modifications on human FEN1 cleavage
Effect of flap modifications on human FEN1 cleavage
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DOI：
10.1021/bi991321u
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发表时间：
1999-10-05
期刊：
影响因子：
2.9
通讯作者：
Bambara, RA
中科院分区：
文献类型：
--
作者：
Bornarth, CJ;Ranalli, TA;Bambara, RA
侧翼核酸内切酶FEN 1在DNA复制和修复中起着关键作用。人FEN 1表现出5'至3'核酸外切活性和结构特异性核酸内切活性。在含有未退火的5 '-尾或瓣结构的引物-模板底物上，FEN 1采用独特的机制在退火点切割，完整地释放S'-尾。FEN 1似乎从5 '端到卵裂点沿着瓣的全长沿着追踪。含有对皮瓣结构修饰的基底已被用于探索追踪机制。为了确定核酸酶是否必须识别瓣上的一系列核苷酸，使用化学接头来替换内部核苷酸。核酸酶可以很容易地穿过这个位点。切割时核酸酶的足迹在瓣上不延伸超过25个核苷酸。11个核苷酸的分支连接到皮瓣以外的足迹区不阻止切割。单-或双-胸腺嘧啶二聚体也允许裂解。受保护区域外的顺式铂加合物具有中度抑制作用。铂改性的分支结构对裂解完全惰性。这些结果表明，一些襟翼修改可以防止或抑制跟踪，但跟踪机制容忍各种襟翼修改。FEN 1有一个灵活的环结构，通过它的皮瓣已被建议线程。然而，分支结构的有效切割与将皮瓣穿过蛋白质中的孔不一致。
The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the S'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.