Related Experiment Video
Updated: Jun 1, 2025
Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Published on: September 11, 2022
Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous
Abstract:
Abasic sites are one of the most frequent forms of DNA damage that interfere with DNA replication. However, abasic sites exhibit complex effects because they can be processed into other types of DNA damage. Thus, it remains poorly understood how abasic sites affect replisome progression, which replication-coupled repair pathways they elicit, and whether this is affected by the template strand that is 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 synthesis of nascent DNA strands but exert different effects when encountered on the leading or lagging strand template. At a leading strand AP site, replisomes stall ∼100 bp from the lesion until it is bypassed or a converging fork triggers termination. At a lagging strand abasic site, replisome progression is unaffected and lagging strands are reprimed downstream, generating a post-replicative gap, which is then bypassed. Despite different effects on replisome progression, both leading and lagging strand abasic sites rely on translesion DNA synthesis for bypass. Our results detail similarities and differences between how leading and lagging strand AP sites affect vertebrate DNA replication.
Insights
Abasic sites stall DNA replication, but their impact differs based on the template strand. Both leading and lagging strand abasic sites are bypassed via translesion DNA synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Abasic sites are common DNA lesions that disrupt DNA replication.
- The precise impact of abasic sites on replisome progression and repair pathways remains unclear.
- Understanding these effects is crucial for comprehending DNA damage tolerance mechanisms.
Purpose of the Study:
- To investigate how abasic sites affect DNA replication fork progression.
- To determine if the template strand influences the processing of abasic sites.
- To identify the DNA repair pathways involved in abasic site bypass.
Main Methods:
- Utilized *Xenopus* egg extracts for in vitro DNA replication studies.
- Employed site-specific, stable abasic sites on leading and lagging strand templates.
- Analyzed replisome stalling, repriming, and gap formation during replication.
Main Results:
- Abasic sites consistently stall nascent DNA strand synthesis.
- Leading strand abasic sites cause replisome stalling until bypass or fork convergence.
- Lagging strand abasic sites lead to repriming and post-replicative gap formation, which is then bypassed.
Conclusions:
- Abasic site processing during replication differs between leading and lagging strands.
- Both leading and lagging strand abasic sites require translesion DNA synthesis for bypass.
- This study elucidates key differences and similarities in vertebrate DNA replication response to abasic sites.
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
02:37Restarting Stalled Replication Forks
02:10Translesion DNA Polymerases
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
02:31Homologous Recombination
02:04Fixing Double-strand Breaks
01:02Long-patch Base Excision Repair