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Updated: Jul 9, 2026
Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle
Published on: December 22, 2023
Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly
Nina Mojas1, Massimo Lopes, Josef Jiricny
1Institute of Molecular Cancer Research, University of Zurich, CH-8057 Zurich, Switzerland.
Abstract:
O(6)-Methylguanine ((Me)G) is a highly cytotoxic DNA modification generated by S(N)1-type methylating agents. Despite numerous studies implicating DNA replication, mismatch repair (MMR), and homologous recombination (HR) in (Me)G toxicity, its mode of action has remained elusive. We studied the molecular transactions in the DNA of yeast and mammalian cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although replication fork progression was unaffected in the first cell cycle after treatment, electron microscopic analysis revealed an accumulation of (Me)G- and MMR-dependent single-stranded DNA (ssDNA) gaps in newly replicated DNA. Progression into the second cell cycle required HR, while the following G(2) arrest required the continued presence of (Me)G. Yeast cells overcame this block, while mammalian cells generally failed to recover, and those that did contained multiple sister chromatid exchanges. Notably, the arrest could be abolished by removal of (Me)G after the first S phase. These new data provide compelling support for the hypothesis that MMR attempts to correct (Me)G/C or (Me)G/T mispairs arising during replication. Due to the persistence of (Me)G in the exposed template strand, repair synthesis cannot take place, which leaves single-stranded gaps behind the replication fork. During the subsequent S phase, these gaps cause replication fork collapse and elicit recombination and cell cycle arrest.
Insights
O(6)-Methylguanine DNA damage triggers replication gaps and cell cycle arrest. Mismatch repair attempts to fix O(6)-Methylguanine, leading to gaps that cause fork collapse and arrest, especially in mammalian cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- O(6)-Methylguanine ((Me)G) is a cytotoxic DNA lesion formed by methylating agents.
- The precise mechanism of (Me)G toxicity involving DNA replication, mismatch repair (MMR), and homologous recombination (HR) remains unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying O(6)-Methylguanine toxicity in yeast and mammalian cells.
- To investigate the roles of DNA replication, MMR, and HR in processing (Me)G lesions.
Main Methods:
- Treatment of yeast and mammalian cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
- Electron microscopy to analyze DNA structures.
- Cell cycle progression analysis.
- Assessment of sister chromatid exchanges.
Main Results:
- Replication fork progression was initially unaffected but led to (Me)G- and MMR-dependent single-stranded DNA gaps in newly replicated DNA.
- Progression into the second cell cycle required HR, and G(2) arrest depended on persistent (Me)G.
- Yeast cells could overcome the arrest, while mammalian cells often failed, exhibiting sister chromatid exchanges.
- Removal of (Me)G after the first S phase abolished the cell cycle arrest.
Conclusions:
- MMR attempts to correct (Me)G/C or (Me)G/T mispairs, but failed repair synthesis leaves ssDNA gaps.
- These gaps cause replication fork collapse and cell cycle arrest in subsequent S phases.
- (Me)G toxicity is mediated by MMR-induced gaps, leading to genomic instability and cell death, particularly in mammalian cells.
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