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Molecular Microbiology Apr 2012Threading of DNA through the central channel of a replicative ring helicase is known as helicase loading, and is a pivotal event during replication initiation at... (Review)
Review
Threading of DNA through the central channel of a replicative ring helicase is known as helicase loading, and is a pivotal event during replication initiation at replication origins. Once loaded, the helicase recruits the primase through a direct protein-protein interaction to complete the initial 'priming step' of DNA replication. Subsequent assembly of the polymerases and processivity factors completes the structure of the replisome. Two replisomes are assembled, one on each strand, and move in opposite directions to replicate the parental DNA during the 'elongation step' of DNA replication. Replicative helicases are the motor engines of replisomes powered by the conversion of chemical energy to mechanical energy through ATP binding and hydrolysis. Bidirectional loading of two ring helicases at a replication origin is achieved by strictly regulated and intricately choreographed mechanisms, often through the action of replication initiation and helicase-loader proteins. Current structural and biochemical data reveal a wide range of different helicase-loading mechanisms. Here we review advances in this area and discuss their implications.
Topics: Bacteria; DNA Helicases; DNA Primase; DNA Replication; DNA, Bacterial; Protein Structure, Tertiary; Replication Origin
PubMed: 22417087
DOI: 10.1111/j.1365-2958.2012.08012.x -
Journal of Bacteriology Aug 2022Nucleoid-associated proteins (NAPs) help structure bacterial genomes and function in an array of DNA transactions, including transcription, recombination, and repair. In...
Nucleoid-associated proteins (NAPs) help structure bacterial genomes and function in an array of DNA transactions, including transcription, recombination, and repair. In most bacteria, NAPs are nonessential in part due to functional redundancy. In contrast, in Bacillus subtilis the HU homolog HBsu is essential for cell viability. HBsu helps compact the B. subtilis chromosome and participates in homologous recombination and DNA repair. However, none of these activities explain HBsu's essentiality. Here, using two complementary conditional HBsu alleles, we investigated the terminal phenotype of the mutants. Our analysis revealed that cells without functional HBsu fail to initiate DNA replication. Importantly, when the chromosomal replication origin () was replaced with a plasmid origin () whose replication does not require the initiator DnaA, cells without HBsu initiated DNA replication normally. However, HBsu was still essential in this -containing strain. We conclude that HBsu plays an essential role in the initiation of DNA replication, likely acting to promote origin melting by DnaA, but also has a second essential function that remains to be discovered. While it is common for a bacterial species to express multiple nucleoid-associated proteins (NAPs), NAPs are seldomly essential for cell survival. In B. subtilis, HBsu is a NAP essential for cell viability. Here, using conditional alleles to rapidly remove or inactivate HBsu, we show that the absence of HBsu abolishes the initiation of DNA replication . Understanding HBsu's function can provide new insights into the regulation of DNA replication initiation in bacteria.
Topics: Bacillus subtilis; Bacterial Proteins; DNA Replication; DNA-Binding Proteins; Replication Origin
PubMed: 35546541
DOI: 10.1128/jb.00119-22 -
ELife Mar 2021Eukaryotic 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...
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.
Topics: Cell Line, Tumor; DNA Replication; Humans; Minichromosome Maintenance Proteins; Models, Genetic; Origin Recognition Complex
PubMed: 33683199
DOI: 10.7554/eLife.62161 -
Nature Communications Nov 2022DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been...
DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.
Topics: Humans; DNA Repair; Genomic Instability; DNA Replication; S Phase; Cell Cycle
PubMed: 36400763
DOI: 10.1038/s41467-022-34379-2 -
Microbiology and Molecular Biology... Mar 2022Plasmids are self-replicative DNA elements that are transferred between bacteria. Plasmids encode not only antibiotic resistance genes but also adaptive genes that allow... (Review)
Review
Plasmids are self-replicative DNA elements that are transferred between bacteria. Plasmids encode not only antibiotic resistance genes but also adaptive genes that allow their hosts to colonize new niches. Plasmid transfer is achieved by conjugation (or mobilization), phage-mediated transduction, and natural transformation. Thousands of plasmids use the rolling-circle mechanism for their propagation (RCR plasmids). They are ubiquitous, have a high copy number, exhibit a broad host range, and often can be mobilized among bacterial species. Based upon the replicon, RCR plasmids have been grouped into several families, the best known of them being pC194 and pUB110 (Rep_1 family), pMV158 and pE194 (Rep_2 family), and pT181 and pC221 (Rep_trans family). Genetic traits of RCR plasmids are analyzed concerning (i) replication mediated by a DNA-relaxing initiator protein and its interactions with the cognate DNA origin, (ii) lagging-strand origins of replication, (iii) antibiotic resistance genes, (iv) mobilization functions, (v) replication control, performed by proteins and/or antisense RNAs, and (vi) the participating host-encoded functions. The mobilization functions include a relaxase initiator of transfer (Mob), an origin of transfer, and one or two small auxiliary proteins. There is a family of relaxases, the MOB family represented by plasmid pMV158, which has been revisited and updated. Family secrets, like a putative open reading frame of unknown function, are reported. We conclude that basic research on RCR plasmids is of importance, and our perspectives contemplate the concept of One Earth because we should incorporate bacteria into our daily life by diminishing their virulence and, at the same time, respecting their genetic diversity.
Topics: Bacterial Proteins; DNA; DNA Replication; DNA, Bacterial; Plasmids
PubMed: 34878299
DOI: 10.1128/MMBR.00222-20 -
Molecular and Cellular Biology Oct 1993We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating...
We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents.
Topics: Animals; CHO Cells; Cricetinae; DNA Replication; Electrophoresis, Gel, Two-Dimensional; Humans; RNA, Ribosomal; Restriction Mapping
PubMed: 8413256
DOI: 10.1128/mcb.13.10.6600-6613.1993 -
Microbiology and Molecular Biology... Mar 2017Bacterial chromosomes initiate replication at a fixed time in the cell cycle, whereas there is generally no particular time for plasmid replication initiation or... (Review)
Review
Bacterial chromosomes initiate replication at a fixed time in the cell cycle, whereas there is generally no particular time for plasmid replication initiation or chromosomal replication initiation from integrated plasmids. In bacteria with divided genomes, the replication system of one of the chromosomes typically resembles that of bacteria with undivided genomes, whereas the remaining chromosomes have plasmid-like replication systems. For example, in Vibrio cholerae, a bacterium with two chromosomes (chromosome 1 [Chr1] and Chr2), the Chr1 system resembles that of the Escherichia coli chromosome, and the Chr2 system resembles that of iteron-based plasmids. However, Chr2 still initiates replication at a fixed time in the cell cycle and thus offers an opportunity to understand the molecular basis for the difference between random and cell cycle-regulated modes of replication. Here we review studies of replication control in Chr2 and compare it to those of plasmids and chromosomes. We argue that although the Chr2 control mechanisms in many ways are reminiscent of those of plasmids, they also appear to combine more regulatory features than are found on a typical plasmid, including some that are more typical of chromosomes. One of the regulatory mechanisms is especially novel, the coordinated timing of replication initiation of Chr1 and Chr2, providing the first example of communication between chromosomes for replication initiation.
Topics: Cell Cycle; Chromosomes, Bacterial; DNA Replication; DNA, Bacterial; Escherichia coli; Gene Dosage; Plasmids; Vibrio cholerae
PubMed: 27903655
DOI: 10.1128/MMBR.00033-16 -
Trends in Immunology Jul 2006Somatic hypermutation (SHM) underlies the generation of a diverse repertoire of high-affinity antibodies. It is effected by a two-step process: (i) DNA lesions initiated... (Review)
Review
Somatic hypermutation (SHM) underlies the generation of a diverse repertoire of high-affinity antibodies. It is effected by a two-step process: (i) DNA lesions initiated by activation-induced cytidine deaminase (AID), and (ii) lesion repair by the combined intervention of DNA replication and repair factors that include mismatch repair (MMR) proteins and translesion DNA synthesis (TLS) polymerases. AID and TLS polymerases that are crucial to SHM, namely polymerase (pol) theta, pol zeta and pol eta, are induced in B cells by the stimuli that are required to trigger this process: B-cell receptor crosslinking and CD40 engagement by CD154. These polymerases, together with MMR proteins and other DNA replication and repair factors, could assemble to form a multimolecular complex ("mutasome") at the site of DNA lesions. Molecular interactions in the mutasome would result in a "polymerase switch", that is, the substitution of the high-fidelity replicative pol delta and pol epsilon with the TLS pol theta, pol eta, Rev1, pol zeta and, perhaps, pol iota, which are error-prone and crucially insert mismatches or mutations while repairing DNA lesions. Here, we place these concepts in the context of the existing in vivo and in vitro findings, and discuss an integrated mechanistic model of SHM.
Topics: Animals; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Escherichia coli; Somatic Hypermutation, Immunoglobulin
PubMed: 16737852
DOI: 10.1016/j.it.2006.05.001 -
Journal of Bacteriology Oct 2008Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a...
Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.
Topics: Bacterial Proteins; Carbon; Caulobacter crescentus; DNA Replication; DNA-Binding Proteins; Gene Deletion; Guanosine Tetraphosphate; Microbial Viability; Pyrophosphatases; Signal Transduction
PubMed: 18723629
DOI: 10.1128/JB.00700-08 -
Proceedings of the National Academy of... Dec 2023DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA...
DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.
Topics: Minichromosome Maintenance Proteins; Saccharomyces cerevisiae Proteins; DNA Replication; DNA; DNA, Single-Stranded
PubMed: 38109526
DOI: 10.1073/pnas.2316466120