Fast and efficient DNA replication with purified human proteins.
Fast and efficient DNA replication with purified human proteins.
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DOI：
10.1038/s41586-022-04759-1
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发表时间：
2022-06
期刊：
影响因子：
64.8
通讯作者：
Yeeles JTP
中科院分区：
文献类型：
--
作者：
Baris Y;Taylor MRG;Aria V;Yeeles JTP
染色体复制是由称为复制体的蛋白质的复杂而复杂的集合体进行的，其中DNA聚合酶Polδ和Polε、DNA聚合酶α-引发酶（Polα）和辅助蛋白包括AND-1、CLASPIN和TIMELESS-TIPIN（在酿酒酵母中分别称为Ctf 4、Mrc 1和Tof 1-Csm 3）围绕CDC 45-MCM-GINS（CMG）复制解旋酶组织。由于功能性的人类复制体尚未从纯化的蛋白质中重建，因此这些因子如何有助于人类DNA复制以及是否需要额外的蛋白质来进行最佳DNA合成尚不清楚。在这里，我们报告的生化重建的人复制体，执行快速和有效的DNA复制使用11个纯化的人复制因子从43多肽。Polε，而不是Polδ，对最佳的前导链合成至关重要。出乎意料的是，Polε介导的前导链复制高度依赖于滑动钳持续合成因子PCNA和替代钳装载复合物CTF 18-RFC。我们展示了CLASPIN和无时间-TIPIN如何促进复制体的进展，并证明了与芽殖酵母复制体相反，AND-1直接增强了前导链复制。此外，尽管AND-1与Polα结合，但这种相互作用对于滞后链复制是无效的，这表明Polα在功能上是通过AND-1非依赖性机制募集的，用于在人类复制体中引发。总的来说，我们的工作揭示了人类复制体如何实现快速有效的前导链和滞后链DNA复制，并为人类复制体及其与其他DNA代谢过程的相互作用的未来研究提供了一个强大的系统。
Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS–TIPIN (respectively known as Ctf4, Mrc1 and Tof1–Csm3 in Saccharomyces cerevisiae) are organized around the CDC45–MCM–GINS (CMG) replicative helicase. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18–RFC. We show how CLASPIN and TIMELESS–TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.
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影响因子：
64.8
作者：
Tunyasuvunakool K;Adler J;Wu Z;Green T;Zielinski M;Žídek A;Bridgland A;Cowie A;Meyer C;Laydon A;Velankar S;Kleywegt GJ;Bateman A;Evans R;Pritzel A;Figurnov M;Ronneberger O;Bates R;Kohl SAA;Potapenko A;Ballard AJ;Romera-Paredes B;Nikolov S;Jain R;Clancy E;Reiman D;Petersen S;Senior AW;Kavukcuoglu K;Birney E;Kohli P;Jumper J;Hassabis D
通讯作者：
Hassabis D
影响因子：
64.8
作者：
通讯作者：
--
DOI：
10.15252/embj.2021108819
发表时间：
2021-12-01
期刊：
The EMBO journal
影响因子：
--
作者：
Jones ML;Baris Y;Taylor MRG;Yeeles JTP
通讯作者：
Yeeles JTP
影响因子：
14.9
作者：
Fujisawa R;Ohashi E;Hirota K;Tsurimoto T
通讯作者：
Tsurimoto T
影响因子：
16
作者：
Taylor MRG;Yeeles JTP
通讯作者：
Yeeles JTP