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Updated: May 29, 2026
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Published on: April 29, 2010
Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork
Andrew D Klocko1, Jeremy W Schroeder, Brian W Walsh
1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.
Insights
Mismatch repair (MMR) corrects DNA replication errors. This study shows MMR proteins like MutS interact with DNA polymerase DnaE, dynamically altering its location during repair to maintain genome stability.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mismatch repair (MMR) is essential for correcting DNA polymerase errors during replication.
- Loss of MMR significantly increases mutation rates, highlighting its critical role in genome maintenance.
- Previous studies suggested a link between MMR and DNA replication machinery in Bacillus subtilis.
Purpose of the Study:
- To investigate the coupling mechanism between MMR and DNA replication.
- To understand the dynamic behavior of MMR and DNA replication proteins in live cells.
- To elucidate the role of MutS in coordinating MMR with DNA synthesis.
Main Methods:
- Utilized live-cell imaging of green fluorescent protein (GFP)-tagged MMR and DNA replication proteins.
- Observed changes in protein foci following induced DNA replication errors.
- Performed in vitro binding assays with MutS, MutL, and DnaE.
Main Results:
- Foci of DNA polymerase DnaE-GFP decreased upon mismatch incorporation, requiring the presence of MutS.
- MutS and MutL were shown to bind DnaE in vitro, indicating a direct interaction.
- DnaE-GFP foci also decreased following DNA synthesis arrest, independent of DNA damage.
Conclusions:
- MMR proteins, particularly MutS, directly interact with the DNA replication machinery (DnaE).
- This interaction causes dynamic reorganization of DnaE at the replication fork during MMR.
- A direct and intimate connection exists between MMR and the replicating DNA polymerase complex in vivo.
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