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.
Dupes
Nicole M
NM
4042 Kraus Natural Science Building, Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
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
D001412:Bacillus subtilis / Q000235:genetics*; D015153:Blotting, Western; D053843:DNA Mismatch Repair / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication / Q000235:genetics*; D008853:Microscopy; D009154:Mutation / Q000235:genetics*; D020125:Mutation, Missense / Q000235:genetics
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