Uncovering the effects of non-lethal oxidative stress on replication initiation in Escherichia coli.

Cell cycle adaptability assists bacteria in response to adverse stress. The effect of oxidative stress on replication initiation in Escherichia coli remains unclear. This work examined the impact of exogenous oxidant and genetic mutation-mediated oxidative stress on replication initiation. We found that 0-0.5 mM H2O2 suppresses E. coli replication initiation in a concentration-dependent manner but does not lead to cell death. Deletion of antioxidant enzymes SodA-SodB, KatE, or AhpC results in delayed replication initiation. The antioxidant N-acetylcysteine (NAC) promotes replication initiation in ΔkatE and ΔsodAΔsodB mutants. We then explored the factors that mediate the inhibition of replication initiation by oxidative stress. MutY, a base excision repair DNA glycosylase, resists inhibition of replication initiation by H2O2. Lon protease deficiency eliminates inhibition of replication initiation mediated by exogenous H2O2 exposure but not by katE or sodA-sodB deletion. The absence of clpP and hslV further delays replication initiation in the ΔktaE mutant, whereas hflK deletion promotes replication initiation in the ΔkatE and ΔsodAΔsodB mutants. In conclusion, non-lethal oxidative stress inhibits replication initiation, and AAA+ proteases are involved and show flexible regulation in E. coli.

Assembly and activation of replicative helicases at origin DNA for replication initiation.

To initiate DNA replication, it is essential to properly assemble a pair of replicative helicases at each replication origin. While the general principle of this process applies universally from prokaryotes to eukaryotes, the specific mechanisms governing origin selection, helicase loading, and subsequent helicase activation vary significantly across different species. Recent advancements in cryo-electron microscopy (cryo-EM) have revolutionized our ability to visualize large protein or protein-DNA complexes involved in the initiation of DNA replication. Complemented by real-time single-molecule analysis, the available high-resolution cryo-EM structures have greatly enhanced our understanding of the dynamic regulation of this process at origin DNA. This review primarily focuses on the latest structural discoveries that shed light on the key molecular machineries responsible for driving replication initiation, with a particular emphasis on the assembly of pre-replication complex (pre-RC) in eukaryotes.

The structural mechanism of dimeric DONSON in replicative helicase activation.

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

Structural insight into H4K20 methylation on H2A.Z-nucleosome by SUV420H1.

DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.

A dual role for the chromatin reader ORCA/LRWD1 in targeting the origin recognition complex to chromatin.

Eukaryotic cells use chromatin marks to regulate the initiation of DNA replication. The origin recognition complex (ORC)-associated protein ORCA plays a critical role in heterochromatin replication in mammalian cells by recruiting the initiator ORC, but the underlying mechanisms remain unclear. Here, we report crystal and cryo-electron microscopy structures of ORCA in complex with ORC's Orc2 subunit and nucleosomes, establishing that ORCA orchestrates ternary complex assembly by simultaneously recognizing a highly conserved peptide sequence in Orc2, nucleosomal DNA, and repressive histone trimethylation marks through an aromatic cage. Unexpectedly, binding of ORCA to nucleosomes prevents chromatin array compaction in a manner that relies on H4K20 trimethylation, a histone modification critical for heterochromatin replication. We further show that ORCA is necessary and sufficient to specifically recruit ORC into chromatin condensates marked by H4K20 trimethylation, providing a paradigm for studying replication initiation in specific chromatin contexts. Collectively, our findings support a model in which ORCA not only serves as a platform for ORC recruitment to nucleosomes bearing specific histone marks but also helps establish a local chromatin environment conducive to subsequent MCM2-7 loading.

Changing protein-DNA interactions promote ORC binding-site exchange during replication origin licensing.

During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC (origin recognition complex) can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA-binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally controlled DNA sliding of helicase-loading intermediates and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA-binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely oriented Mcm2-7 helicases to ensure bidirectional DNA replication.

Involvement of OxyR and Dps in the repression of replication initiation by DsrA small RNA in Escherichia coli.

Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas ß-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.

Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Loss of Ezh2 function remodels the DNA replication initiation landscape.

In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.

The human pre-replication complex is an open complex.

In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

DDK promotes DNA replication initiation: Mechanistic and structural insights.

DNA replication initiation in eukaryotes is tightly regulated through two cell-cycle specific processes, replication licensing to install inactive minichromosome maintenance (MCM) double-hexamers (DH) on origins in early G1 phase and origin firing to assemble and activate Cdc45-Mcm2-7-GINS (CMG) helicases upon S phase entry. Two kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are responsible for driving the association of replication factors with the MCM-DH to form CMG helicases for origin melting and DNA unwinding and eventually replisomes for bi-directional DNA synthesis. In recent years, cryo-electron microscopy studies have generated a collection of structural snapshots for the stepwise assembly and remodeling of the replication initiation machineries, creating a framework for understanding the regulation of this fundamental process at a molecular level. Very recent progress is the structural characterization of the elusive MCM-DH-DDK complex, which provides insights into mechanisms of kinase activation, substrate recognition and selection, as well as molecular role of DDK-mediated MCM-DH phosphorylation in helicase activation.

Regulation of DNA replication initiation by ParA is independent of parS location in Bacillus subtilis.

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis, the Par system (ParA/Soj, ParB/Spo0J and parS) is required for segregation of the chromosome origin (oriC) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC. Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS.

Link Between Antibiotic Persistence and Antibiotic Resistance in Bacterial Pathogens.

Both, antibiotic persistence and antibiotic resistance characterize phenotypes of survival in which a bacterial cell becomes insensitive to one (or even) more antibiotic(s). However, the molecular basis for these two antibiotic-tolerant phenotypes is fundamentally different. Whereas antibiotic resistance is genetically determined and hence represents a rather stable phenotype, antibiotic persistence marks a transient physiological state triggered by various stress-inducing conditions that switches back to the original antibiotic sensitive state once the environmental situation improves. The molecular basics of antibiotic resistance are in principle well understood. This is not the case for antibiotic persistence. Under all culture conditions, there is a stochastically formed, subpopulation of persister cells in bacterial populations, the size of which depends on the culture conditions. The proportion of persisters in a bacterial population increases under different stress conditions, including treatment with bactericidal antibiotics (BCAs). Various models have been proposed to explain the formation of persistence in bacteria. We recently hypothesized that all physiological culture conditions leading to persistence converge in the inability of the bacteria to re-initiate a new round of DNA replication caused by an insufficient level of the initiator complex ATP-DnaA and hence by the lack of formation of a functional orisome. Here, we extend this hypothesis by proposing that in this persistence state the bacteria become more susceptible to mutation-based antibiotic resistance provided they are equipped with error-prone DNA repair functions. This is - in our opinion - in particular the case when such bacterial populations are exposed to BCAs.

Transcription shapes DNA replication initiation to preserve genome integrity.

BACKGROUND: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. RESULTS: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. CONCLUSION: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

G-quadruplex DNA and RNA: Their roles in regulation of DNA replication and other biological functions.

G-quadruplex is one of the best-studied non-B type DNA that is now known to be prevalently present in the genomes of almost all the biological species. Recent studies reveal roles of G-quadruplex (G4) structures in various nucleic acids and chromosome transactions. In this short article, we will first describe recent findings on the roles of G4 in regulation of DNA replication. G4 is involved in regulation of spatio-temporal regulation of DNA replication through interaction with a specific binding protein, Rif1. This regulation is at least partially mediated by generation of specific chromatin architecture through Rif1-G4 interactions. We will also describe recent studies showing the potential roles of G4 in initiation of DNA replication. Next, we will present showcases of highly diversified roles of DNA G4 and RNA G4 in regulation of nucleic acid and chromosome functions. Finally, we will discuss how the formation of cellular G4 could be regulated.

Positive and Negative Regulation of DNA Replication Initiation.

Genomic DNA is replicated every cell cycle by the programmed activation of replication origins at specific times and chromosomal locations. The factors that define the locations of replication origins and their typical activation times in eukaryotic cells are poorly understood. Previous studies highlighted the role of activating factors and epigenetic modifications in regulating replication initiation. Here, we review the role that repressive pathways - and their alleviation - play in establishing the genomic landscape of replication initiation. Several factors mediate this repression, in particular, factors associated with inactive chromatin. Repression can support organized, yet stochastic, replication initiation, and its absence could explain instances of rapid and random replication or re-replication.

Regulation of DNA Binding and High-Order Oligomerization of the DnaB Helicase Loader.

DnaB is an essential primosomal protein that coloads the replicative helicase in many Gram-positive bacteria, including several human pathogens. Although DnaB is tetrameric in solution, it is from a protein family whose members can oligomerize into large complexes when exposed to DNA. It is currently unknown how DNA binding by DnaB is regulated or how these interactions induce changes in its oligomeric state. Here, we investigated DNA binding by DnaB from Bacillus subtilis and the critical human pathogen Staphylococcus aureus We found that B. subtilis DnaB binds double-stranded DNA as a tetramer; however, M13mp18 single-stranded DNA induces high-order oligomerization. Mutating a conserved motif at the C-terminal end of DnaB stimulates single-stranded DNA binding, suggesting that conformational changes in this region regulate DNA substrate preferences. S. aureus DnaB could also be induced to form high-order oligomers with either M13mp18 or PhiX174 single-stranded DNA. Therefore, oligomeric shifts in DnaB are tightly controlled and this activity is conserved between B. subtilis and a pathogenic species.IMPORTANCE DnaB is a replicative helicase loader involved in initiating DNA replication in many bacterial species. We investigated the binding preferences of DnaB for its DNA substrate and determined that the C-terminal end of the protein plays a critical role in controlling DNA interactions. Furthermore, we found that DNA binding in general did not trigger changes to the oligomeric state of DnaB, but rather, certain types of single-stranded DNA substrates specifically induced DnaB to self-assemble into a large complex. This indicates that the structure of DNA itself is an important regulatory element that influences the behavior of DnaB. Importantly, these observations held for both Bacillus subtilis and the pathogenic species Staphylococcus aureus, demonstrating conserved biochemical functions of DnaB in these species.

Structural ensemble and biological activity of DciA intrinsically disordered region.

DciA is a newly discovered bacterial protein involved in loading the replicative helicase DnaB onto DNA at the initiation step of chromosome replication. Its three-dimensional structure is composed of a folded N-terminal domain (residues 1-111) resembling K Homology domains and a long disordered C-terminal tail (residues 112-157) which structure-activity relationship remains to be elucidated. In the present study on Vibrio cholerae DciA, we emphasize the importance of its disordered region to load DnaB onto DNA using surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). Then we characterize the conformational ensemble of the full-length protein using a combination of circular dichroism (CD), small angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. The atomic-level structural ensemble generated by MD simulations is in very good agreement with SAXS data. From initial conformations of the C-terminal tail without any secondary structure, our simulations bring to light several transient helical structures in this segment, which might be molecular recognition features (MoRFs) for the binding to DnaB and its recruitment and loading onto DNA.

Persistence of Intracellular Bacterial Pathogens-With a Focus on the Metabolic Perspective.

Persistence has evolved as a potent survival strategy to overcome adverse environmental conditions. This capability is common to almost all bacteria, including all human bacterial pathogens and likely connected to chronic infections caused by some of these pathogens. Although the majority of a bacterial cell population will be killed by the particular stressors, like antibiotics, oxygen and nitrogen radicals, nutrient starvation and others, a varying subpopulation (termed persisters) will withstand the stress situation and will be able to revive once the stress is removed. Several factors and pathways have been identified in the past that apparently favor the formation of persistence, such as various toxin/antitoxin modules or stringent response together with the alarmone (p)ppGpp. However, persistence can occur stochastically in few cells even of stress-free bacterial populations. Growth of these cells could then be induced by the stress conditions. In this review, we focus on the persister formation of human intracellular bacterial pathogens, some of which belong to the most successful persister producers but lack some or even all of the assumed persistence-triggering factors and pathways. We propose a mechanism for the persister formation of these bacterial pathogens which is based on their specific intracellular bipartite metabolism. We postulate that this mode of metabolism ultimately leads, under certain starvation conditions, to the stalling of DNA replication initiation which may be causative for the persister state.