小柯机器人

英国MRC分子生物学实验室Joseph T. P. Yeeles研究团队开发出利用纯化人类蛋白质进行快速高效DNA复制的方法。相关论文于2022年5月18日发表在《自然》杂志上。

他们报告了一种人类复制体的生化重组方法，其使用由 43 种多肽制成的 11 种纯化的人类复制因子进行快速高效 DNA 复制。Polε，但不是 Polδ，对于最佳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加工性因子 PCNA 和替代钳装载机复合物 CTF18-RFC。他们展示了 CLAPIN 和 TIMELESS-TIPIN 如何促进复制体的进展，并证明与出芽酵母复制体相比，AND-1 直接增强了前导链复制。此外，虽然 AND-1 与 聚合酶 α-引发酶 (Polα)结合，但这种相互作用对于滞后链复制来说是可有可无的，这表明 Polα 在功能上是通过一种与 AND-1 无关的机制在人类复制体中引发的。总的来说，他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，并为未来研究人类复制体及其与其他 DNA 代谢过程的相互作用提供了一个强大的系统。

据了解，染色体复制由称为复制体的复杂蛋白质集合进行，其中 DNA 聚合酶 Polδ 和 Polε、DNA Polα和辅助蛋白包括 AND-1、CLASPIN 和 TIMELESS-TIPIN（分别称为 Ctf4 , 酿酒酵母中的 Mrc1 和 Tof1–Csm3) 围绕 CDC45–MCM–GINS (CMG) 复制解旋酶组织。由于尚未从纯化的蛋白质中重建功能性人类复制体，因此对这些因素如何促进人类 DNA 复制以及最佳 DNA 合成是否需要额外的蛋白质知之甚少。

附：英文原文

Title: Fast and efficient DNA replication with purified human proteins

Author: Baris, Yasemin, Taylor, Martin R. G., Aria, Valentina, Yeeles, Joseph T. P.

Issue&Volume: 2022-05-18

Abstract: 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 helicase1,2,3,4,5,6,7. 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 replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, 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.

DOI: 10.1038/s41586-022-04759-1

Source: https://www.nature.com/articles/s41586-022-04759-1

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
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