Kao Hui-I, Veeraraghavan Janaki, Polaczek Piotr, Campbell Judith L, Bambara Robert A
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
J Biol Chem. 2004 Apr 9;279(15):15014-24. doi: 10.1074/jbc.M313216200. Epub 2004 Jan 26.
Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.
被称为冈崎片段的短DNA片段是真核生物DNA复制的中间体。每个片段都包含一个引发RNA/DNA引物（iRNA/DNA），该引物会转化为5'-翼瓣，然后在片段连接之前被去除。在这个过程的一种模型中，翼瓣内切核酸酶1（FEN1）去除iRNA。在另一种模型中，单链结合蛋白复制蛋白A（RPA）覆盖翼瓣，抑制FEN1，但刺激Dna2p解旋酶/核酸酶的切割作用。RPA从产生的短翼瓣上解离，从而使FEN1进行切割。为了确定最可能的过程，我们分析了短和长5'-翼瓣的切割情况。FEN1能在一个高效反应中切割10个核苷酸的固定或平衡翼瓣，即使在高水平的RPA或Dna2p存在时也不受影响。对于30个核苷酸的固定或平衡翼瓣，RPA会部分抑制FEN1。CTG翼瓣可形成回折结构，对两种核酸酶都有抑制作用，然而，在CTG翼瓣的5'-末端添加一个dT(12)可使Dna2p进行切割。在有利于解旋酶活性的反应条件下，高活性的Dna2p能显著刺激带尾回折翼瓣以及30个核苷酸的无结构翼瓣的FEN1切割。我们的结果表明，大多数翼瓣的加工不使用Dna2p。然而，Dna2p在结构化翼瓣的加工途径中发挥作用，它利用其核酸酶和解旋酶活性协助FEN1。
