Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes

Sara Rodriguez-Acebes,Silvana Mourón,Juan Méndez

doi:10.1074/jbc.ra118.003740

Sara Rodriguez-Acebes, Silvana Mourón + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ra118.003740

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Abstract

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes “replicative stress” that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.

Highlights

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the generation
The protein machinery responsible for new DNA synthesis is recruited to thousands of replication origins in the G1 phase of the cell division cycle
Experimental conditions were chosen for which the primary effect could be inferred beforehand

Summary

Edited by Patrick Sung

DNA replication precedes mitotic cell division to transmit genetic information to the generation. To as “fork speed” or “fork rate” (FR).3 Both parameters can be analyzed at the single-molecule level using DNA combing or stretched chromatin fibers in cells sequentially labeled with nucleotide analogues CldU and IdU [1, 2]. Accurate IOD estimations require long and stable DNA fibers, and a practical alternative is to quantify the number of origins activated during the CldU pulse (referred to as “first-label origins”) relative to a fixed number of total replication structures, including origins, forks, and termination events [4]. Replication forks are slowed down and occasionally stopped as they encounter special DNA structures, transcription proteins, or DNA lesions introduced by radiation or toxic chemicals This phenomenon is referred to as “replicative stress” (RS) and is normally counteracted by mechanisms that protect stalled forks and promote the restart of DNA synthesis (9 –12). Alterations that slow down forks trigger the activation of otherwise “dormant” origins as a compensatory mechanism [19, 20], whereas alterations that primarily increase the number of origins lead to slower forks because the additional

Cause and effect in replicative stress

Results and discussion

Cell culture and manipulations

Immunoblots and antibodies

Flow cytometry detection of IdU incorporation and DNA content