Conti Brooke A, Ruiz Penelope D, Broton Cayla, Blobel Nicolas J, Kottemann Molly C, Sridhar Sunandini, Lach Francis P, Wiley Tom, Sasi Nanda K, Carroll Thomas, Smogorzewska Agata
Laboratory of Genome Maintenance, The Rockefeller University.
Laboratory for Cell Biology and Genetics, The Rockefeller University.
bioRxiv. 2023 Mar 13:2023.03.13.532415. doi: 10.1101/2023.03.13.532415.
Genetic information is duplicated via the highly regulated process of DNA replication. The machinery coordinating this process, the replisome, encounters many challenges, including replication fork-stalling lesions that threaten the accurate and timely transmission of genetic information. Cells have multiple mechanisms to repair or bypass lesions that would otherwise compromise DNA replication. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2) function to regulate Replication Termination Factor 2 (RTF2) at the stalled replisome, allowing for replication fork stabilization and restart. Here we show that RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes. We show that during unperturbed DNA replication, RTF2, like RNase H2, is required to maintain normal replication fork speeds. However, persistent RTF2 and RNase H2 at stalled replication forks compromises the replication stress response, preventing efficient replication restart. Such restart is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for regulation of replication-coupled ribonucleotide incorporation during normal replication and the replication stress response that is achieved through RTF2. We also provide evidence for PRIM1 function in direct replication restart following replication stress in mammalian cells.
遗传信息通过高度调控的DNA复制过程进行复制。协调这一过程的机制——复制体，面临着许多挑战，包括威胁遗传信息准确及时传递的复制叉停滞损伤。细胞具有多种机制来修复或绕过那些否则会损害DNA复制的损伤。我们之前已经表明，蛋白酶体穿梭蛋白DNA损伤诱导因子1和2（DDI1/2）在停滞的复制体处发挥作用，调节复制终止因子2（RTF2），从而实现复制叉的稳定和重启。在此我们表明，RTF2调节核糖核酸酶H2在复制体中的定位，核糖核酸酶H2是一种异源三聚体酶，负责在RNA-DNA异源双链体的背景下去除RNA。我们发现，在正常的DNA复制过程中，与核糖核酸酶H2一样，RTF2是维持正常复制叉速度所必需的。然而，在停滞的复制叉处持续存在的RTF2和核糖核酸酶H2会损害复制应激反应，阻碍高效的复制重启。这种重启依赖于PRIM1，即DNA聚合酶α-引发酶的引发酶成分。我们的数据表明，在正常复制以及通过RTF2实现的复制应激反应过程中，对复制偶联的核糖核苷酸掺入进行调控至关重要。我们还为PRIM1在哺乳动物细胞复制应激后的直接复制重启中的作用提供了证据。
