RESUMO
During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.
Assuntos
Bacillus subtilis/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Bacteriano/biossíntese , Peptidoglicano/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Dano ao DNA/genética , DNA Bacteriano/genética , Proteínas de Membrana/genética , Peptidoglicano/metabolismoRESUMO
Homologous recombination requires the coordinated effort of several proteins to complete break resection, homologous pairing, and resolution of DNA crossover structures. RecN is a conserved bacterial protein important for double-strand break repair and is a member of the structural maintenance of chromosomes (SMC) protein family. Current models in Bacillus subtilis propose that RecN responds to double-stranded breaks prior to RecA and end processing, suggesting that RecN is among the very first proteins responsible for break detection. Here, we investigate the contribution of RecA and end processing by AddAB to RecN recruitment into repair foci in vivo. Using this approach, we found that recA is required for RecN-green fluorescent protein (GFP) focus formation on the nucleoid during normal growth and in response to DNA damage. In the absence of recA function, RecN foci form in a low percentage of cells, RecN localizes away from the nucleoid, and RecN fails to assemble in response to DNA damage. In contrast, we show that the response of RecA-GFP foci to DNA damage is unchanged in the presence or absence of recN. In further support of RecA activity preceding RecN, we show that ablation of the double-strand break end-processing enzyme addAB results in a failure of RecN to form foci in response to DNA damage. With these results, we conclude that RecA and end processing function prior to RecN, establishing a critical step for the recruitment and participation of RecN during DNA break repair in Bacillus subtilis. IMPORTANCE Homologous recombination is important for the repair of DNA double-strand breaks. RecN is a highly conserved protein that has been shown to be important for sister chromatid cohesion and for surviving break-inducing clastogens. Here, we show that the assembly of RecN into repair foci on the bacterial nucleoid requires the end-processing enzyme AddAB and the recombinase RecA. In the absence of either recA or end-processing RecN-GFP, foci are no longer DNA damage inducible, and foci form in a subset of cells as large complexes in regions away from the nucleoid. Our results establish the stepwise order of action, where double-strand break end processing and RecA association precede the participation of RecN in break repair in Bacillus subtilis.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Reparo do DNA , Enzimas de Restrição do DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Recombinases Rec A/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Dano ao DNA , Enzimas de Restrição do DNA/genética , DNA Bacteriano , Genótipo , Recombinases Rec A/genéticaRESUMO
RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/genética , Uracila/análogos & derivados , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Mutação , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão , Uracila/farmacologiaRESUMO
During mismatch repair, MutS is responsible for mismatch detection and the recruitment of MutL to the mismatch through a mechanism that is unknown in most organisms. Here, we identified a discrete site on MutS that is occupied by MutL in Bacillus subtilis. The MutL binding site is composed of two adjacent phenylalanine residues located laterally in an exposed loop of MutS. Disruption of this site renders MutS defective in binding MutLâ in vitro and in vivo, while also eliminating mismatch repair. Analysis of MutS repair complexes in vivo shows that MutS mutants defective in interaction with MutL are 'trapped' in a repetitive loading response. Furthermore, these mutant MutS repair complexes persist on DNA away from the DNA polymerase, suggesting that MutS remains loaded on mismatch proximal DNA awaiting arrival of MutL. We also provide evidence that MutS and MutL interact independent of mismatch binding by MutSâ in vivo and in vitro, suggesting that MutL can transiently probe MutS to determine if MutS is mismatch bound. Together, these data provide insights into the mechanism that MutS employs to recruit MutL, and the consequences that ensue when MutL recruitment is blocked.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Reparo de Erro de Pareamento de DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Evolução Molecular , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Taxa de Mutação , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de Sinais/genéticaRESUMO
Mismatch repair (MMR) increases the fidelity of DNA replication by identifying and correcting replication errors. Processivity clamps are vital components of DNA replication and MMR, yet the mechanism and extent to which they participate in MMR remains unclear. We investigated the role of the Bacillus subtilis processivity clamp DnaN, and found that it serves as a platform for mismatch detection and coupling of repair to DNA replication. By visualizing functional MutS fluorescent fusions in vivo, we find that MutS forms foci independent of mismatch detection at sites of replication (i.e. the replisome). These MutS foci are directed to the replisome by DnaN clamp zones that aid mismatch detection by targeting the search to nascent DNA. Following mismatch detection, MutS disengages from the replisome, facilitating repair. We tested the functional importance of DnaN-mediated mismatch detection for MMR, and found that it accounts for 90% of repair. This high dependence on DnaN can be bypassed by increasing MutS concentration within the cell, indicating a secondary mode of detection in vivo whereby MutS directly finds mismatches without associating with the replisome. Overall, our results provide new insight into the mechanism by which DnaN couples mismatch recognition to DNA replication in living cells.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Reparo de Erro de Pareamento de DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismoRESUMO
Mismatch repair is a highly conserved pathway responsible for correcting DNA polymerase errors incorporated during genome replication. MutL is a mismatch repair protein known to coordinate several steps in repair that ultimately results in strand removal following mismatch identification by MutS. MutL homologs from bacteria to humans contain well-conserved N-terminal and C-terminal domains. To understand the contribution of the MutL N-terminal domain to mismatch repair, we analyzed 14 different missense mutations in Bacillus subtilis MutL that were conserved with missense mutations identified in the human MutL homolog MLH1 from patients with hereditary nonpolyposis colorectal cancer (HNPCC). We characterized missense mutations in or near motifs important for ATP binding, ATPase activity, and DNA binding. We found that 13 of the 14 missense mutations conferred a substantial defect to mismatch repair in vivo, while three mutant alleles showed a dominant negative increase in mutation frequency to wild-type mutL. We performed immunoblot analysis to determine the relative stability of each mutant protein in vivo and found that, although most accumulated, several mutant proteins failed to maintain wild-type levels, suggesting defects in protein stability. The remaining missense mutations located in areas of the protein important for DNA binding, ATP binding, and ATPase activities of MutL compromised repair in vivo. Our results define functional residues in the N-terminal domain of B. subtilis MutL that are critical for mismatch repair in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Pareamento Incorreto de Bases/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Instabilidade Genômica , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
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.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Reparo de Erro de Pareamento de DNA , DNA Polimerase III/metabolismo , Replicação do DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , DNA Polimerase III/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Bacillus subtilis/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Replicação do DNA/genética , Mutação/genética , Western Blotting , Reparo de Erro de Pareamento de DNA/genética , Microscopia , Mutação de Sentido Incorreto/genéticaRESUMO
DNA mismatch repair (MMR) is responsible for correcting errors formed during DNA replication. DNA polymerase errors include base mismatches and extra helical nucleotides referred to as insertion and deletion loops. In bacteria, MMR increases the fidelity of the chromosomal DNA replication pathway approximately 100-fold. MMR defects in bacteria reduce replication fidelity and have the potential to affect fitness. In mammals, MMR defects are characterized by an increase in mutation rate and by microsatellite instability. In this review, we discuss current advances in understanding how MMR functions in bacteria lacking the MutH and Dam methylase-dependent MMR pathway.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Bactérias Gram-Positivas/genética , Adenosina Trifosfatases/metabolismo , Animais , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Aptidão Genética , Bactérias Gram-Positivas/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , MutaçãoRESUMO
From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.
Assuntos
Bacillus subtilis/genética , Reparo do DNA , Replicação do DNA , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Dano ao DNA , Mutagênese , Esporos Bacterianos/fisiologiaRESUMO
Protein-protein interactions are required for the proper function of many biological pathways. Numerous biochemical and protein blotting methods are available for probing direct and indirect interactions between two protein-binding partners. Here, we describe the methodology of far Western blotting, or immunodot blotting, as a technique for probing direct interactions between two proteins. We describe the utility of this approach as a rapid, qualitative screen for identifying novel protein-binding partners. We also describe the importance of this technique for measuring differences in interaction between wild-type and mutant forms of a known binding partner. Far Western blotting is a rapid and highly reproducible experimental approach for identifying and understanding the interaction between protein-binding partners leading to new discoveries in the function and regulation of biological pathways.
Assuntos
Reparo do DNA , Replicação do DNA , Mapeamento de Interação de Proteínas/métodos , Far-Western Blotting/métodos , Biologia Molecular/métodos , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Fatores de TempoRESUMO
Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.