Related Experiment Video
Updated: Feb 9, 2026
Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response
Published on: August 2, 2024
Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass
Zhenyu Zou1, Ze Chen1, Qizhen Xue1
1Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, No.17 People's South Road, Chengdu, 6100041, P. R. China.
Abstract:
The DNA replisome inevitably encounters DNA damage during DNA replication. The T7 DNA replisome contains a DNA polymerase (gp5), the processivity factor thioredoxin (trx), a helicase-primase (gp4), and a ssDNA-binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated strand-displacement DNA synthesis past 8-oxoG or O6 -MeG lesions at the synthetic DNA fork by the T7 DNA replisome. DNA damage does not obviously affect the binding affinities between helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6 -MeG-as well as GC-rich template sequence clusters-inhibit strand-displacement DNA synthesis and produce partial extension products. Relative to the gp4 ΔC-tail, gp4 promotes DNA damage bypass. The presence of gp2.5 also promotes it. Thus, the interactions of polymerase with helicase and ssDNA-binding protein facilitate DNA damage bypass. Accessory proteins in other complicated DNA replisomes also facilitate bypassing DNA damage in similar manner. This work provides new mechanistic information relating to DNA damage bypass by the DNA replisome.
Insights
The T7 DNA replisome
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA replication is essential for life, but the DNA replisome frequently encounters DNA damage.
- The T7 DNA replisome comprises DNA polymerase (gp5), thioredoxin (trx), helicase-primase (gp4), and ssDNA-binding protein (gp2.5).
- The role of protein interactions in DNA damage bypass by the T7 replisome remains under-explored.
Purpose of the Study:
- To investigate how T7 replisome protein interactions facilitate DNA damage bypass during replication.
- To examine strand-displacement DNA synthesis past 8-oxoG and O6-MeG lesions at a synthetic DNA fork.
Main Methods:
- Utilized a synthetic DNA fork model to study T7 DNA replisome activity.
- Assessed the impact of DNA lesions (8-oxoG, O6-MeG) on DNA synthesis and protein-DNA binding.
- Compared the bypass capabilities of wild-type gp4 and a gp4 variant lacking the C-tail, with and without gp2.5.
Main Results:
- DNA damage (8-oxoG, O6-MeG) and GC-rich sequences inhibited strand-displacement synthesis, leading to partial extension products.
- DNA damage did not significantly alter binding affinities between replisome proteins and the DNA fork.
- Wild-type gp4 and the presence of gp2.5 significantly promoted DNA damage bypass compared to controls.
Conclusions:
- Interactions between the T7 DNA polymerase, helicase, and ssDNA-binding protein are crucial for facilitating DNA damage bypass.
- These findings suggest that accessory proteins in other DNA replisomes may similarly enhance DNA damage tolerance.
- This study provides novel mechanistic insights into DNA damage bypass during replication.
Related Concept Videos
03:01The Replisome
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
03:06From DNA to Protein
02:36DNA Damage Can Stall the Cell Cycle
02:36DNA Damage can Stall the Cell Cycle
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...
00:55DNA Helicases