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Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation
Eugen C Minca1, David Kowalski
1Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Abstract:
The integrity of the genome is threatened by DNA damage that blocks the progression of replication forks. Little is known about the genomic locations of replication fork stalling, and its determinants and consequences in vivo. Here we show that bulky DNA damaging agents induce localized fork stalling at yeast replication origins, and that localized stalling is dependent on proximal origin activity and is modulated by the intra-S-phase checkpoint. Fork stalling preceded the formation of sister chromatid junctions required for bypassing DNA damage. Despite DNA adduct formation, localized fork stalling was abrogated at an origin inactivated by a point mutation and prominent stalling was not detected at naturally-inactive origins in the replicon. The intra-S-phase checkpoint contributed to the high-level of fork stalling at early origins, while checkpoint inactivation led to initiation, localized stalling and chromatid joining at a late origin. Our results indicate that replication forks initially encountering a bulky DNA adduct exhibit a dual nature of stalling: a checkpoint-independent arrest that triggers sister chromatid junction formation, as well as a checkpoint-enhanced arrest at early origins that accompanies the repression of late origin firing. We propose that the initial checkpoint-enhanced arrest reflects events that facilitate fork resolution at subsequent lesions.
Insights
Replication forks stall at DNA damage sites, influenced by origin activity and the intra-S-phase checkpoint. This stalling precedes DNA repair mechanisms like sister chromatid junction formation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA damage threatens genome integrity by blocking replication fork progression.
- The precise genomic locations, determinants, and in vivo consequences of replication fork stalling remain largely unknown.
Purpose of the Study:
- To investigate the genomic localization and regulation of replication fork stalling in yeast.
- To elucidate the role of origin activity and the intra-S-phase checkpoint in fork stalling and DNA damage bypass.
Main Methods:
- Utilized yeast as a model organism to study DNA damage response.
- Employed techniques to monitor replication fork progression and stalling in vivo.
- Investigated the impact of origin inactivation and checkpoint modulation on fork stalling.
Main Results:
- Bulky DNA damaging agents induce localized replication fork stalling at yeast replication origins.
- Stalling is dependent on proximal origin activity and modulated by the intra-S-phase checkpoint.
- Fork stalling precedes sister chromatid junction formation for DNA damage bypass; stalling is abrogated at inactivated origins.
Conclusions:
- Replication fork stalling at DNA adducts has a dual nature: checkpoint-independent arrest triggering sister chromatid junction formation and checkpoint-enhanced arrest at early origins.
- The intra-S-phase checkpoint plays a critical role in repressing late origin firing and enhancing fork stalling at early origins.
- These findings provide insights into the mechanisms of genome maintenance and DNA damage tolerance during replication.
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