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oai:repisalud.isciii.es:20.500.12105/83662024-11-29T13:58:39Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597

00925njm 22002777a 4500 dc Rodriguez-Acebes, Sara author Mouron, Silvana Andrea author Mendez, Juan author 2018-08-17 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. J Biol Chem. 2018;293(33):12855-12861 0021-9258 http://hdl.handle.net/20.500.12105/8366 29959228 10.1074/jbc.RA118.003740 1083-351X The Journal of biological chemistry DNA polymerase DNA primase DNA replication Cell division cycle 7-related protein kinase (Cdc7) Fork speed Molecular biology Replication origin Replicative stress Stretched DNA fibers Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes