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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., (© 2018 Rodriguez-Acebes et al.)

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)