Rossi Marie L, Pike Jason E, Wang Wensheng, Burgers Peter M J, Campbell Judith L, Bambara Robert A
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 2008 Oct 10;283(41):27483-27493. doi: 10.1074/jbc.M804550200. Epub 2008 Aug 9.
Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.
真核冈崎片段成熟需要在连接发生之前完全去除起始RNA引物。聚合酶δ（Pol δ）延伸上游冈崎片段，并将下游引物的5'端置换为单核苷酸侧翼，该侧翼通过FEN1核酸酶切割去除。这个过程重复进行，直到所有RNA都被去除。然而，一小部分侧翼逃避了切割并生长得足够长，以至于被RPA覆盖，并且需要Dna2和FEN1核酸酶的连续作用来进行处理。在这里，我们测试了RPA是否如所提出的那样抑制长侧翼的FEN1切割。令人惊讶的是，我们确定RPA与由聚合酶δ动态形成的长侧翼结合仅略微抑制FEN1切割，显然消除了对Dna2的需求。因此，我们询问其他相关蛋白质是否通过Dna2途径促进长侧翼切割。从遗传学研究中可知，参与冈崎片段成熟的Pif1解旋酶改善了侧翼置换，并增加了RPA对FEN1切割长侧翼的抑制作用。这些结果表明，Pif1加速了长侧翼的生长，使RPA在FEN1发挥作用之前就能够结合，从而抑制FEN1切割。因此，Pif1将长侧翼导向双核酸酶途径，需要Dna2切割来去除引物。
