2024-03-29T10:59:52Zhttp://repisalud.isciii.es/oai/requestoai:repisalud.isciii.es:20.500.12105/83662023-10-09T12:08:35Zcom_20.500.12105_2174com_20.500.12105_2051com_20.500.12105_2173col_20.500.12105_2175
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