Control of DNA replication in vitro using a reversible replication barrier.

A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-ß-D-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1-2 weeks and experiments using this approach are typically performed over 1-3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials.

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination.

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.