2C-RT Quantifies Genome-Wide DNA Replication Perturbationsand Redefines RIF1-CDC7 Regulation of Replication Timing
- 1Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden
Abstract
Replication timing (RT) coordinates DNA synthesis with genome organization and influences mutation rates, cancer evolution, and developmental plasticity. Current methods infer S-phase timing from DNA content, an assumption that obscures responses to replication perturbations and limits accurate comparisons of RT changes. Here, we develop 2C-RT, a multiplexed sequencing approach that uses DNA content-independent markers for robust RT profiling across S phase. Combining 2C-RT with imaging, we resolve distinct layers of human RT control and identify local and genome-wide responses to drugs and genetic perturbations. We find that RIF1 spatially restricts CDC7 activity and that its loss does not neutralize RT per se but advances replication across most RIF1-sensitive regions. A conserved MCM5 bob1 mutation lowers the origin-activation threshold and reveals a role for CDC7 in establishing local origin dominance. These findings support a revised model in which CDC7 primes early origins, CDK promotes permissive origin firing genome-wide, and RIF1 counteracts CDC7 in distinct chromatin states and peripheral nuclear domains.
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BibTeX
@misc{urien20262crt,
title = {{2C-RT Quantifies Genome-Wide DNA Replication Perturbationsand Redefines RIF1-CDC7 Regulation of Replication Timing}},
author = {B. Urien and P. Y. Kuwong and G. Giglio and C. Navarro and M. Rodrigues da Fonseca and S. Pradhan and S. Elsasser and B. Lemmens},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.758048},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.758048v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - 2C-RT Quantifies Genome-Wide DNA Replication Perturbationsand Redefines RIF1-CDC7 Regulation of Replication Timing
AU - B. Urien
AU - P. Y. Kuwong
AU - G. Giglio
AU - C. Navarro
AU - M. Rodrigues da Fonseca
AU - S. Pradhan
AU - S. Elsasser
AU - B. Lemmens
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.758048
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.758048v1
AB - Replication timing (RT) coordinates DNA synthesis with genome organization and influences mutation rates, cancer evolution, and developmental plasticity. Current methods infer S-phase timing from DNA content, an assumption that obscures responses to replication perturbations and limits accurate comparisons of RT changes. Here, we develop 2C-RT, a multiplexed sequencing approach that uses DNA content-independent markers for robust RT profiling across S phase. Combining 2C-RT with imaging, we resolve distinct layers of human RT control and identify local and genome-wide responses to drugs and genetic perturbations. We find that RIF1 spatially restricts CDC7 activity and that its loss does not neutralize RT per se but advances replication across most RIF1-sensitive regions. A conserved MCM5 bob1 mutation lowers the origin-activation threshold and reveals a role for CDC7 in establishing local origin dominance. These findings support a revised model in which CDC7 primes early origins, CDK promotes permissive origin firing genome-wide, and RIF1 counteracts CDC7 in distinct chromatin states and peripheral nuclear domains.
ER -