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In JoVE (1)
Other Publications (17)
- Molecular Microbiology
- Molecular Microbiology
- Molecular Microbiology
- The Journal of Biological Chemistry
- The EMBO Journal
- Proceedings of the National Academy of Sciences of the United States of America
- Molecular Microbiology
- Molecular Cell
- Journal of Bacteriology
- Journal of Bacteriology
- Molecular Microbiology
- Molecular Cell
- Journal of Bacteriology
- Molecular Cell
- Molecular Microbiology
- PLoS Pathogens
- Molecular Microbiology
Articles by Lyle A. Simmons in JoVE
Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis
Andrew D. Klocko, Kaleena M. Crafton, Brian W. Walsh, Justin S. Lenhart, Lyle A. Simmons
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 Lyle A. Simmons on PubMed
The DnaAcos Allele of Escherichia Coli: Hyperactive Initiation is Caused by Substitution of A184V and Y271H, Resulting in Defective ATP Binding and Aberrant DNA Replication Control
Molecular Microbiology. Feb, 2003 | Pubmed ID: 12535074
Chromosomal DNA replication is regulated at the level of commitment to this biochemical pathway. In Escherichia coli, DnaA protein appears to regulate this process. A mutant form, DnaAcos, carrying four amino acid substitutions, is apparently defective in responding to regulatory signals, because it induces hyperactive initiation from the bacterial replication origin (oriC). In this report, the phenotype of hyperactive initiation is shown to be the result of two specific amino acid substitutions. One (A184V) immediately adjacent to a Walker A box (P loop motif) causes a defect in ATP binding (Carr and Kaguni, 1996, Mol Microbiol 20: 1307-1318). The second amino acid substitution (Y271H) appears to stabilize the activity of the mutant protein carrying the A184V substitution. The mutant protein carrying both amino acid substitutions (A184V + Y271H) is defective in modulating the frequency of initiation from oriC, as demonstrated by marker frequency analysis of oriC and a locus near the replication terminus. These results indicate that a defect in ATP binding results in aberrant control of DNA replication.
DnaA Protein of Escherichia Coli: Oligomerization at the E. Coli Chromosomal Origin is Required for Initiation and Involves Specific N-terminal Amino Acids
Molecular Microbiology. Aug, 2003 | Pubmed ID: 12864864
Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.
Hyperinitiation of DNA Replication in Escherichia Coli Leads to Replication Fork Collapse and Inviability
Molecular Microbiology. Jan, 2004 | Pubmed ID: 14756777
Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.
An Essential Tryptophan of Escherichia Coli DnaA Protein Functions in Oligomerization at the E. Coli Replication Origin
The Journal of Biological Chemistry. Jul, 2005 | Pubmed ID: 15878847
In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.
Y-family DNA Polymerases Respond to DNA Damage-independent Inhibition of Replication Fork Progression
The EMBO Journal. Feb, 2006 | Pubmed ID: 16482223
In Escherichia coli, the Y-family DNA polymerases Pol IV (DinB) and Pol V (UmuD2'C) enhance cell survival upon DNA damage by bypassing replication-blocking DNA lesions. We report a unique function for these polymerases when DNA replication fork progression is arrested not by exogenous DNA damage, but with hydroxyurea (HU), thereby inhibiting ribonucleotide reductase, and bringing about damage-independent DNA replication stalling. Remarkably, the umuC122::Tn5 allele of umuC, dinB, and certain forms of umuD gene products endow E. coli with the ability to withstand HU treatment (HUR). The catalytic activities of the UmuC122 and DinB proteins are both required for HUR. Moreover, the lethality brought about by such stalled replication forks in the wild-type derivatives appears to proceed through the toxin/antitoxin pairs mazEF and relBE. This novel function reveals a role for Y-family polymerases in enhancing cell survival under conditions of nucleotide starvation, in addition to their established functions in response to DNA damage.
Proceedings of the National Academy of Sciences of the United States of America. Jan, 2007 | Pubmed ID: 17229847
In both prokaryotes and eukaryotes, proteins involved in DNA repair often organize into multicomponent complexes that can be visualized as foci in living cells. We used a RecA-GFP fusion to examine the subcellular cues that direct RecA-GFP to assemble as foci in response to DNA damage. We used two different methods to inhibit initiation of DNA replication and determined that DNA replication is required for the cell to establish RecA-GFP foci after exposure to DNA-damaging agents. Furthermore, use of endonuclease cleavage to generate a site-specific double-strand break demonstrated that the replication machinery (replisome) and DNA synthesis are required for assembly of RecA-GFP foci during repair of a double-strand break. We monitored the cellular levels of RecA and found that focus formation does not require further induction of protein levels, suggesting that foci result from a redistribution of existing protein to sites of damage encountered by the replisome. Taken together, our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. These results provide insight into the mechanisms that the cell uses to recruit repair proteins to damaged DNA in living cells.
Multiple Ku Orthologues Mediate DNA Non-homologous End-joining in the Free-living Form and During Chronic Infection of Sinorhizobium Meliloti
Molecular Microbiology. Jan, 2008 | Pubmed ID: 18067541
The bacterial non-homologous end-joining (NHEJ) apparatus is a two-component system that uses Ku and LigD to repair DNA double-strand breaks. Although the reaction mechanism has been extensively studied, much less is known about the physiological role of bacterial NHEJ. Recent studies suggest that NHEJ acts under conditions where DNA replication is reduced or absent (such as in a spore or stationary phase). Interestingly, genes encoding Ku and LigD have been identified in a wide range of bacteria that can chronically infect eukaryotic hosts. Strikingly, Sinohizobium meliloti, an intracellular symbiont of legume plants, carries four genes encoding Ku homologues (sku1 to sku4). Deletion analysis of the sku genes indicated that all Ku homologues are functional. One of these genes, sku2, is strongly expressed in free-living cells, as well as in bacteroid cells residing inside of the host plant. To visualize the NHEJ apparatus in vivo, SKu2 protein was fused to yellow fluorescent protein (YFP). Ionizing radiation (IR) induced focus formation of SKu2-YFP in free-living cells in a dosage-dependent manner. Moreover, SKu2-YFP foci formed in response to IR in non-dividing bacteroids, indicating that NHEJ system is functional even during the chronic infection phase of symbiosis.
Molecular Cell. Feb, 2008 | Pubmed ID: 18280235
MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an interaction with beta clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of the beta clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with beta clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and beta is the major molecular interaction facilitating focus formation and that beta clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with beta clamp.
Journal of Bacteriology. Oct, 2008 | Pubmed ID: 18689473
Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.
Comparison of Responses to Double-strand Breaks Between Escherichia Coli and Bacillus Subtilis Reveals Different Requirements for SOS Induction
Journal of Bacteriology. Feb, 2009 | Pubmed ID: 19060143
DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.
Sinorhizobium Meliloti CpdR1 is Critical for Co-ordinating Cell Cycle Progression and the Symbiotic Chronic Infection
Molecular Microbiology. Aug, 2009 | Pubmed ID: 19602145
ATP-driven proteolysis plays a major role in regulating the bacterial cell cycle, development and stress responses. In the nitro -fixing symbiosis with host plants, Sinorhizobium meliloti undergoes a profound cellular differentiation, including endoreduplication of the ome. The regulatory mechanisms governing the alterations of the S. meliloti cell cycle in planta are largely unknown. Here, we report the characterization of two cpdR homologues, cpdR1 and cpdR2, of S. meliloti that encode single-domain response regulators. In Caulobacter crescentus, CpdR controls the polar localization of the ClpXP protease, thereby mediating the regulated proteolysis of key protein(s), such as CtrA, involved in cell cycle progression. The S. meliloti cpdR1-null mutant can invade the host cytoplasm, however, the intracellular bacteria are unable to differentiate into bacteroids. We show that S. meliloti CpdR1 has a polar localization pattern and a role in ClpX positioning similar to C. crescentus CpdR, suggesting a conserved function of CpdR proteins among alpha-proteobacteria. However, in S. meliloti, free-living cells of the cpdR1-null mutant show a striking morphology of irregular coccoids and aberrant DNA replication. Thus, we demonstrate that CpdR1 mediates the co-ordination of cell cycle events, which are critical for both the free-living cell division and the differentiation required for the chronic intracellular infection.
Molecular Cell. Dec, 2009 | Pubmed ID: 20005847
Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. Although HU inhibition of RNR is well recognized, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death, we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability. Continued HU stress activates iron uptake and toxins MazF and RelE, whose activity causes the synthesis of incompletely translated proteins and stimulation of envelope stress responses. These effects alter the properties of one of the cell's terminal cytochrome oxidases, causing an increase in superoxide production. The increased superoxide production, together with the increased iron uptake, fuels the formation of hydroxyl radicals that contribute to HU-induced cell death.
Mutations in the Bacillus Subtilis Beta Clamp That Separate Its Roles in DNA Replication from Mismatch Repair
Journal of Bacteriology. Jul, 2010 | Pubmed ID: 20453097
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.
Molecular Cell. Jul, 2010 | Pubmed ID: 20603082
DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.
Molecular Microbiology. Oct, 2010 | Pubmed ID: 20807199
The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5'- and 3'-ends of 16S rRNA as well as maturation of the 5'-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.
DNA Damage and Reactive Nitrogen Species Are Barriers to Vibrio Cholerae Colonization of the Infant Mouse Intestine
PLoS Pathogens. Feb, 2011 | Pubmed ID: 21379340
Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.
Molecular Microbiology. Nov, 2011 | Pubmed ID: 21958350
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.