Murante R S, Henricksen L A, Bambara R A
Department of Biochemistry and Biophysics, and Cancer Center, Box 712, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2244-9. doi: 10.1073/pnas.95.5.2244.
The initiator RNAs of mammalian Okazaki fragments are thought to be removed by RNase HI and the 5'-3' flap endonuclease (FEN1). Earlier evidence indicated that the cleavage site of RNase HI is 5' of the last ribonucleotide at the RNA-DNA junction on an Okazaki substrate. In current work, highly purified calf RNase HI makes this exact cleavage in Okazaki fragments containing mismatches that distort the hybrid structure of the heteroduplex. Furthermore, even fully unannealed Okazaki fragments were cleaved. Clearly, the enzyme recognizes the transition from RNA to DNA on a single-stranded substrate and not the RNA/DNA heteroduplex structure. We have named this junction RNase activity. This activity exactly comigrates with RNase HI activity during purification strongly suggesting that both activities reside in the same enzyme. After junction cleavage, FEN1 removes the remaining ribonucleotide. Because FEN1 prefers a substrate with a single-stranded 5'-flap structure, the single-stranded activity of junction RNase suggests that Okazaki fragments are displaced to form a 5'-tail prior to cleavage by both nucleases.
哺乳动物冈崎片段的起始RNA被认为是由核糖核酸酶HI和5'-3'翼状内切核酸酶（FEN1）去除的。早期证据表明，核糖核酸酶HI的切割位点位于冈崎底物上RNA-DNA连接处最后一个核糖核苷酸的5'端。在当前的工作中，高度纯化的小牛核糖核酸酶HI在含有错配的冈崎片段中进行了精确切割，这些错配会扭曲异源双链体的杂交结构。此外，即使是完全未退火的冈崎片段也能被切割。显然，该酶识别单链底物上从RNA到DNA的转变，而不是RNA/DNA异源双链体结构。我们将这种连接核糖核酸酶活性命名为。在纯化过程中，这种活性与核糖核酸酶HI活性完全共迁移，强烈表明这两种活性存在于同一种酶中。连接切割后，FEN1去除剩余的核糖核苷酸。由于FEN1更喜欢具有单链5'-翼状结构的底物，连接核糖核酸酶的单链活性表明冈崎片段在被两种核酸酶切割之前会被置换形成5'-尾。
