JBC:北京大学孔道春实验室阐明真核细胞岗崎片段加工成熟的分子
近日，国际知名生物化学杂志《Journal of BioLogical Chemistry》在线发表了北京大学生命科学学院孔道春教授研究组的一篇研究论文，研究论文报道了一种体内直接检测去除RNA-DNA引物的方法。
岗崎片段(Okazaki fragments)的发现已经过去50多年了，但真核细胞岗崎片段加工的分子机制一直没有得到最终确定。人细胞的一次生长分裂，大约合成5 x 107岗崎片段。一个岗崎片段的平均大小约是125 nt，它的5'端含有～34核苷酸的RNA-DNA引物。这段引物是由低保真的primase-DNA pol α 合成。为了能让岗崎片段连接起来形成一个连续的后随DNA链(lagging strand)，这个RNA-DNA引物必须要从每一个岗崎片段里切除掉。所以，岗崎片段加工是细胞里DNA transactions最丰富的事件，而且它直接关系到基因组的稳定性。
为什么真核细胞岗崎片段加工的分子机制一直不能被确定呢?主要有两个原因：1)缺乏能在体内直接检测去除～34核苷酸的RNA-DNA引物的方法;2)应该有多个酶参与去除RNA-DNA引物，它们之间存在一定程度的功能互补，导致无法明确地确定哪些酶参与岗崎片段加工。以前，岗崎片段加工的分子机制研究主要是在体外进行，再加上一些有限的遗传学分析。经过30年的研究，科研人员提出了可能存在的加工岗崎片段的途径，但不同实验室有非常不同的观点。由于缺乏体内的直接证据，真核细胞去除岗崎片段上RNA-DNA引物的分子机制一直无法最终确定(Kao & Bambara (2003) The protein components and mechanism of eukaryotic Okazaki fragment maturation. Crit. Rev. Biochem. Mol. Biol.38, 433-452)。
研究组首次用电镜观察到了复制叉里的翘起结构(flap structure)，证明了翘起结构的存在;并进一步证明这个翘起结构来自于RNA-DNA引物。他们证明了真核细胞用两种方法去除RNA-DNA引物：Flap切割途径(Flap pathway)和外切酶途径(Exonucleolytic pathway)。Flap pathway可再分为long flap pathway及short flap pathway，他们进一步确定Dna2、Fen1、Exo1、RNase H2参与了这些途径。由此，真核细胞岗崎片段加工成熟的分子机制得到最终阐明。
岗崎片段加工机制的模型图
原文链接：
Direct Visualization of RNA-DNA primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells
原文摘要：
During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo. In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo. The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.
作者：孔道春
