Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801
Proc Natl Acad Sci U S A. 2019 Jan 22;116(4):1251-1260. doi: 10.1073/pnas.1814512116. Epub 2019 Jan 7.
In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficient comprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficient generates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication in is effectively semidiscontinuous.
在体外，纯化的复制体驱动模型复制叉合成连续的前导链，即使没有连接酶，也支持 DNA 复制的半不连续模型。然而，从缺乏连接酶的细胞中分离出的新生复制中间体仅包含短的（平均 1.2kb）冈崎片段。长期以来，人们一直怀疑细胞通过体外观察到的半不连续模式复制其染色体 DNA，但在体内，新生的前导链在合成后通过切除修复被人为地碎片化。在这里，我们使用脉冲标记的复制中间体的高分辨率分离与链特异性杂交相结合，表明具有切除修复能力的 产生的前导链中间体比滞后链冈崎片段长 10 倍以上。DNA 修复活性（包括核苷酸切除）的失活进一步将新生前导链的大小增加到约 80kb，而滞后链冈崎片段不受影响。我们得出结论，在体内，引发在先导链上的发生频率比滞后链低约 70 倍，并且 在 中 DNA 复制实际上是半不连续的。
