Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (2)
Articles by Justin S. Lenhart in JoVE
JoVE General
Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis
Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor
A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.
Other articles by Justin S. Lenhart on PubMed
Mutations in the Bacillus Subtilis Beta Clamp That Separate Its Roles in DNA Replication from Mismatch Repair
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.
Mismatch Repair Causes the Dynamic Release of an Essential DNA Polymerase from the Replication Fork
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.
