Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate
Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate
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DOI：
10.1074/jbc.m110662200
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发表时间：
2002-04-26
影响因子：
4.8
通讯作者：
Bambara, RA
中科院分区：
文献类型：
--
作者：
Kao, KI;Henricksen, LA;Bambara, RA
Flap内切核酸酶1（FEN 1）是一种结构特异性核酸酶，其在冈崎片段加工期间切割含有未退火的5 '-flap的底物。解理在退火点或退火点附近去除瓣片。古细菌FEN 1或细菌DNA聚合酶的5 ′-核酸酶结构域的优选底物是双瓣结构，其在邻近5 ′-瓣的上游引物上含有3 ′-尾。我们报告，FEN 1在酿酒酵母（Rad 27 p）表现出类似的特异性。与传统测试的完全退火的上游引物相比，当上游引物含有1个核苷酸的3 '-尾时，切割最有效。切割位点仅在退火区域中的一个核苷酸的位置处，允许人DNA连接酶I密封所有产生的切口。相比之下，来自传统翼片基底的一部分产物未被结扎。上游引物的3 '-OH对于双瓣识别不是关键的，因为Rad 27 p耐受修饰。然而，3 '-核苷酸的定位限定了切割位点。我们已经测试了具有互补尾部的底物，所述互补尾部通过分支迁移与许多结构平衡。FEN 1仅切割含有1个核苷酸3 '-尾的那些。在5 '瓣末端平衡含有12-核糖核苷酸的底物模拟体内情况。Rad 27 p以预期的1-核苷酸3 '-尾构型切割该底物。总之，这些结果表明，双瓣基板的形成和切割过程中真核DNA在体内复制。
Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5'-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN 1 or the 5'-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3'-tail on the upstream primer adjacent to the 5'-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3'-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3'-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3'-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5'-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3'-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.