Replication fork uncoupling causes nascent strand degradation and fork reversal

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicati...

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Published inNature structural & molecular biology Vol. 30; no. 1; pp. 115 - 124
Main Authors Kavlashvili, Tamar, Liu, Wenpeng, Mohamed, Taha M, Cortez, David, Dewar, James M
Format Journal Article
LanguageEnglish
Published United States Nature Publishing Group 01.01.2023
Subjects
Animals
Degradation
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA helicase
DNA Helicases - metabolism
DNA polymerase
DNA Replication
DNA-Binding Proteins - metabolism
Genomes
Genomic Instability
Humans
Molecular biology
Replication
Replication forks
Xenopus laevis - metabolism
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Summary:Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal.
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WL performed the experiments in Fig. 2d, Fig. 6h, Extended Data Fig. 1b, Extended Data Fig. 3j,k, and Extended Data Fig. 4f, Extended Data Fig. 8f. TM performed the experiments in Fig. 3h, Extended Data Fig. 1c. TK performed all other experiments. TK and JMD designed the experiments, analyzed the data, and wrote the paper with input from DC, TM, and WL.
AUTHOR CONTRIBUTIONS
ISSN:1545-9993
1545-9985
DOI:10.1038/s41594-022-00871-y