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Mutations in the Bacillus subtilis beta clamp that separate its roles in DNA replication from mismatch repair.

ABSTRACT: The beta clamp is an essential replication sliding clamp required for processive DNA synthesis. The beta clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of beta clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49 degrees C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49 degrees C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the beta clamp, a common site occupied by proteins that bind the beta clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis beta clamp separate the role of the beta clamp in DNA replication from its role in MMR.

SUBMITTER: Dupes NM

PROVIDER: S-EPMC2897676 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Mutations in the Bacillus subtilis beta clamp that separate its roles in DNA replication from mismatch repair.

Dupes Nicole M NM Walsh Brian W BW Klocko Andrew D AD Lenhart Justin S JS Peterson Heather L HL Gessert David A DA Pavlick Cassie E CE Simmons Lyle A LA

Journal of bacteriology 20100507 13

The beta clamp is an essential replication sliding clamp required for processive DNA synthesis. The beta clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of beta clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49 degrees C, and they show an increase in mutation frequency caused by a ...[more]

PMID: 20453097

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Project description:UnlabelledEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.ImportanceHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

Dataset Information

Mutations in the Bacillus subtilis beta clamp that separate its roles in DNA replication from mismatch repair.

Publications

Mutations in the Bacillus subtilis beta clamp that separate its roles in DNA replication from mismatch repair.

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