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Evidence that MutSβ repairs indels generated by mispair initiated template slippage
Scott A Lujan1, Alan B Clark1, Jessica S Williams1
1Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Abstract:
This study examines the specificity of MutSβ dependent DNA mismatch repair (MMR) at the URA3 reporter gene in Saccharomyces cerevisiae. Addition and deletion mutations containing multiple base changes are observed at elevated rates in msh3∆ strains. This is consistent with a role for MutSβ in repairing multibase mismatches, such as those important in development of certain diseases and possibly including those made during Okazaki fragment maturation. Mutations containing single C-G base pair deletions are also observed at elevated rates in msh3∆ strains. These mutations are observed at still higher rates in msh3∆ strains with mutator variants of the three major replicases, implicating a defect in insertion/deletion repair during nuclear DNA replication. These deletions are observed at only a subset of detectable locations in URA3 and they occur at higher rates in one of two orientations of URA3 relative to the nearest replication origin. This subset of sites has a strong local DNA sequence context bias suggesting that the deletions are initiated by transient base•base mismatches which lead to template strand slippage, a type of transient initiator mutagenesis (TIM). We conclude that these mispair-initiated template slippage events require MMR via MutSβ before the next round of DNA synthesis to prevent deletion of an unpaired cytosine in the template strand.
Insights
MutSβ (MutS beta) DNA mismatch repair (MMR) is crucial for fixing errors during DNA replication. This study shows MutSβ prevents deletions caused by template strand slippage, particularly single base deletions, highlighting its role in maintaining genome stability.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- DNA mismatch repair (MMR) corrects errors during DNA replication.
- MutSβ is a key component of the MMR system, particularly involved in repairing insertion-deletion loops.
- Understanding MMR specificity is vital for comprehending genome stability and disease development.
Purpose of the Study:
- To investigate the specificity of MutSβ-dependent MMR at the URA3 reporter gene in Saccharomyces cerevisiae.
- To determine the role of MutSβ in repairing multibase and single-base deletions.
- To elucidate the mechanism of deletion formation and its dependence on DNA replication and sequence context.
Main Methods:
- Utilized the URA3 reporter gene system in Saccharomyces cerevisiae.
- Compared mutation rates in wild-type and msh3∆ strains (lacking MutSβ).
- Introduced mutator variants of replicases to assess their impact on mutation rates.
Main Results:
- Elevated rates of multibase and single-base deletions were observed in msh3∆ strains.
- Single-base deletions were further increased in msh3∆ strains with mutator replicases, implicating replication fidelity.
- Deletion hotspots exhibited sequence context bias and orientation dependence relative to replication origins, suggesting transient initiator mutagenesis (TIM).
Conclusions:
- MutSβ plays a critical role in repairing multibase mismatches and preventing single-base deletions.
- Template strand slippage, initiated by transient mismatches, is a significant source of deletions.
- MMR by MutSβ is essential to prevent the loss of unpaired cytosines during DNA replication.
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