Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

Seco, Elena M.; Ayora, Silvia

doi:10.1093/nar/gkx493

Cited by 24 publications

(34 citation statements)

References 62 publications

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“…HU proteins from various organisms have been found to form homo-or heterodimers 193 [18,19,22,53]. To determine the oligomeric state of C. difficile HupA protein, we performed 194 size exclusion chromatography [60]. The elution profile of the purified protein was compared 195 to molecular weight standards on a Superdex 75 HR 10/30 column.…”

Section: Disruption Of Dna Binding Does Not Affect Oligomerization 192mentioning

confidence: 99%

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

Paiva

Friggen

Qin

et al. 2018

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26The maintenance and organization of the chromosome plays an important role in the 27 development and survival of bacteria. Bacterial chromatin proteins are architectural proteins 28 that bind DNA, modulate its conformation and by doing so affect a variety of cellular 29 processes. No bacterial chromatin proteins of C. difficile have been characterized to date. 30Here, we investigate aspects of the C. difficile HupA protein, a homologue of the 31 histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a 32homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. 33 difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA 34 binding, HupA induces a conformational change in the substrate DNA in vitro and leads to 35 compaction of the chromosome in vivo. 36The present study is the first to characterize a bacterial chromatin protein in C. 37 difficile and opens the way to study the role of chromosomal organization in DNA 38 metabolism and on other cellular processes in this organism. 39 survival [24, 25]. However, in B. subtilis the HU protein HBsu is essential for cell viability, 67 likely due to the lack of functional redundancy of the HU proteins such as in E. coli [17, 23]. 68In solution, most HU proteins are found as homodimers or heterodimers and are able 69 to bind DNA through a flexible DNA binding domain. The crystal structure of the E. coli αHU-70 βHU heterodimer suggests the formation of higher order complexes at higher protein 71 concentrations [22]. Modeling of these complexes suggest HU proteins have the ability to 72 form higher-order complexes through dimer-dimer interaction and make nucleoprotein 73 filaments [22, 26, 27]. However, the physiological relevance of these is still unclear [18, 22, 74 27]. 75The flexible nature of the DNA-binding domain in HU proteins confers the ability to 76 accommodate diverse substrates. Most proteins bind with variable affinity and without strong 77 sequence specificity to both DNA and RNA [28]. Some bacterial chromatin proteins have a 78 clear preference for AT-rich regions [29][30][31] or for the presence of different structures on the 79 DNA [28, 32]. 80 HU proteins can modulate DNA topology in various ways. They can stabilize 81 negatively supercoiled DNA or constrain negative supercoils in the presence of 82 topoisomerase [22, 33]. HU proteins are involved in modulation of the chromosome 83 conformation and have been shown to compact DNA [16, 26, 34]. This compaction of DNA is 84 possible through the ability of HU proteins to introduce flexible hinges and/or bend the DNA 85 [16, 26, 34, 35]. 86The ability to induce conformational changes in the DNA influences a variety of 87 cellular processes due to an indirect effect on global gene expression [36][37][38][39][40]. In E. coli HU 88 proteins are differentially expressed during the cell cycle. The αHU-βHU heterodimer is 89 prevalent in stationary phase, while during exponential growth HU is predominantly present 90 a...

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Section: Disruption Of Dna Binding Does Not Affect Oligomerization 192mentioning

confidence: 99%

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

Paiva

Friggen

Qin

et al. 2018

Preprint

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“…The SPP1 replication and packaging machineries have been studied in deep ( Alonso et al, 2006 ; Lo Piano et al, 2011 ; Oliveira et al, 2013 ). SPP1 DNA replication starts by the theta mode when the replisome organizer, G 38 P, binds to the replication origin, ori L ( Pedre et al, 1994 ; Missich et al, 1997 ; Seco and Ayora, 2017 ). Then, the phage helicase loader (G 39 P) recruits the replicative haxameric helicase (G 40 P).…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1

et al. 2017

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Natural transformation and viral-mediated transduction are the main avenues of horizontal gene transfer in Firmicutes. Bacillus subtilis SPP1 is a generalized transducing bacteriophage. Using this lytic phage as a model, we have analyzed how viral replication and recombination systems contribute to the transfer of plasmid-borne antibiotic resistances. Phage SPP1 DNA replication relies on essential phage-encoded replisome organizer (G38P), helicase loader (G39P), hexameric replicative helicase (G40P), recombinase (G35P) and in less extent on the partially dispensable 5′→3′ exonuclease (G34.1P), the single-stranded DNA binding protein (G36P) and the Holliday junction resolvase (G44P). Correspondingly, the accumulation of linear concatemeric plasmid DNA, and the formation of transducing particles were blocked in the absence of G35P, G38P, G39P, and G40P, greatly reduced in the G34.1P, G36P mutants, and slightly reduced in G44P mutants. In contrast, establishment of injected linear plasmid DNA in the recipient host was independent of viral-encoded functions. DNA homology between SPP1 and the plasmid, rather than a viral packaging signal, enhanced the accumulation of packagable plasmid DNA. The transfer efficiency was also dependent on plasmid copy number, and rolling-circle plasmids were encapsidated at higher frequencies than theta-type replicating plasmids.

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“…Importantly, DNA replication must respond to situations of changing environmental or developmental conditions, including response to damage in the template, and reduced nutritional capacity of cells (5). This is especially relevant for bacterial cells that are directly affected by changes in their surroundings (6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

“…We have sought to shed light onto the question how Bacillus subtilis, a model organism especially for the large group of Gram-positive bacteria, adjusts replication at a single molecule level, in response to amino acid starvation. Interestingly, the architecture of B. subtilis replication forks is rather similar to that of eukaryotic cells, and dissimilar to other model bacteria such as E. coli, in that two replicative polymerases act at the lagging strand(43,(52)(53)(54), besides other differences.Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s)(9). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub.…”

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confidence: 96%

Single molecule dynamics at a bacterial replication fork after nutritional downshift

Hernández-Tamayo

Schmitz

Graumann

2020

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Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and of lagging strand polymerase DnaE in the model bacterium Bacillus subtilis in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability, we show that proteins react differentially to the stress conditions. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after arrest of polymerase PolC, but binds much less often after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone ppGpp due to the stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behaviour of DnaG and of DnaE suggest that both proteins are recruited independently to the forks, rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.

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Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

Cited by 24 publications

References 62 publications

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1

Single molecule dynamics at a bacterial replication fork after nutritional downshift

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