title: Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork.
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.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Klocko
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Schroeder
authors.forename: Brian W
authors.initials: BW
authors.lastname: Walsh
authors.forename: Justin S
authors.initials: JS
authors.lastname: Lenhart
authors.forename: Margery L
authors.initials: ML
authors.lastname: Evans
authors.forename: Lyle A
authors.initials: LA
authors.lastname: Simmons
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000502:physiology*; D053843:DNA Mismatch Repair; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D051719:MutS DNA Mismatch-Binding Protein