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Updated: Jan 16, 2026
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous
Matthew T Cranford1, Steven N Dahmen1, David Cortez1
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
Abstract:
Abasic sites are frequent DNA lesions that interfere with replication and exert complex biological effects because they can be processed into other lesions. Thus, it remains poorly understood how abasic sites affect replisome progression, which repair pathways they elicit, and whether this depends on the template strand damaged. Using Xenopus egg extracts, we developed an approach to analyze replication of DNA containing a site-specific, stable abasic site on the leading or lagging strand template. We show that abasic sites robustly stall DNA synthesis but exert strand-specific effects. Leading strand abasic sites stall leading strands at the lesion, while lagging strands stall downstream at template-dependent positions. We conclude that replisomes uncouple at leading strand lesions, then stall due to additional template constraints. Synthesis restarts upon lesion bypass or when a converging fork triggers termination. In contrast, lagging strand abasic sites stall only lagging strands, indicating replisome progression was unaffected. Lagging strands reprime downstream, generating a post-replicative gap that is subsequently filled. Despite different effects on replisome progression, both leading and lagging strand abasic sites require translesion DNA synthesis for bypass. Our results reveal how strand-specific abasic sites differentially affect replication and demonstrate that uncoupled replisomes are susceptible to downstream template constraints.
Insights
Abasic sites stall DNA replication in a strand-specific manner. Leading strand lesions halt replication forks, while lagging strand lesions cause downstream stalls, both requiring translesion synthesis for bypass.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Abasic sites are common DNA lesions impacting DNA replication and cellular function.
- The precise mechanisms by which abasic sites affect DNA replisome progression and the influence of the damaged template strand remain unclear.
Purpose of the Study:
- To investigate how site-specific abasic sites on either the leading or lagging strand template influence DNA replication.
- To elucidate the strand-specific effects of abasic sites on replisome progression and repair pathway activation.
Main Methods:
- Utilized Xenopus egg extracts to analyze DNA replication in the presence of a stable, site-specific abasic site.
- Developed an experimental approach to differentiate the effects on leading versus lagging strand synthesis.
Main Results:
- Abasic sites consistently stall DNA synthesis, exhibiting distinct strand-specific behaviors.
- Leading strand abasic sites cause replication forks to stall directly at the lesion.
- Lagging strand abasic sites induce downstream stalls on the lagging strand, with leading strand progression unaffected, and necessitate repriming and gap filling.
Conclusions:
- Replisomes uncouple at leading strand abasic sites, subsequently stalling due to template constraints.
- Lagging strand abasic sites lead to repriming and post-replicative gap formation without affecting overall replisome progression.
- Both leading and lagging strand abasic sites require translesion DNA synthesis for replication bypass, highlighting its critical role in lesion tolerance.
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