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Accelerated growth in the absence of DNA replication origins

ABSTRACT: DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life1 and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whilst most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We have used deep sequencing to study replication in Haloferax volcanii. Four chromosomal origins of differing activity were identified. Deletion of individual origins resulted in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild-type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved. Measurement of replication dynamics (marker frequency analysis; MFA) for Haloferax volcanii strains, including wild-type, the laboratory strain, individual and combinations of replication origin deletions.

ORGANISM(S): Haloferax volcanii

SUBMITTER: Conrad Nieduszynski

PROVIDER: E-GEOD-41961 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Accelerated growth in the absence of DNA replication origins

Project description:DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life1 and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whilst most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We have used deep sequencing to study replication in Haloferax volcanii. Four chromosomal origins of differing activity were identified. Deletion of individual origins resulted in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild-type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

2013-11-03 | GSE41961 | GEO

Checking the origin utilisation of Haloferax mediterranei H13 through high-throughput DNA sequencing

Project description:A potential origin that appears to stay dormant in its native host Haloferax volcanii lacking the main active origins becomes activated and competent for replication of the entire chromosome when integrated into the chromosome of the origin-deleted H. mediterranei. Measurement of replication dynamics (marker frequency analysis; MFA) for Haloferax mediterranei H13

2015-07-10 | E-GEOD-70592 | biostudies-arrayexpress

Evolution of genome architecture in Archaea: spontaneous generation of a new chromosome in Haloferax volcanii by homologous recombination

Project description:Eukaryotic genomes typically consist of multiple (linear) chromosomes that are replicated from multiple origins. Several hypothetical scenarios have been proposed to account for the evolution of multi-origin/multi-chromosome genomes, which are encountered in modern eukaryotes and archaea. Here we report an example of the generation of a new chromosome in the halophilic archaeon Haloferax volcanii through one of these scenarios: acquisition of new replication origins and splitting of an ancestral chromosome into two replication-competent chromosomes. The multi-origin main chromosome has split into two genome elements via homologous recombination. The newly generated elements possess all the features of bona fide chromosomes. To our knowledge, the spontaneous generation of a new chromosome in prokaryotes without horizontal gene transfer has not been reported previously.

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Checking the origin utilisation of Haloferax mediterranei H13 through high-throughput DNA sequencing

2015-07-10 | GSE70592 | GEO

Avoiding chromosome pathology when replication forks collide

Project description:Chromosome duplication normally initiates via the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to trigger re-replication of the already replicated DNA, thus posing a threat to genomic integrity. In Escherichia coli this threat is kept at bay by the RecG DNA translocase. Without RecG, replication initiates where forks meet, establishing new forks with the potential to sustain cell growth and division in the absence of an active origin. The studies reported raise the question of how eukaryotic and archaeal cells are able to exploit multiple origins for the duplication of each chromosome without any apparent ill effect from the consequent multiple fork collisions. Measurement of replication dynamics (marker frequency analysis; MFA) for E. coli strains, including wild-type and various mutants.

2013-07-22 | E-GEOD-41975 | biostudies-arrayexpress

The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog (Mnase-Seq)

Project description:We use MNase-Seq to elucidate primary chromatin architecture in an archaeon without histones, the acido-thermophilic archaeon Thermoplasma acidophilum. Like all members of the Thermoplasmatales, T. acidophilum harbours a HU family protein, HTa, that is highly expressed and protects - like histones but unlike well-characterized bacterial HU proteins – a sizeable fraction of the genome from MNase digestion. Comparing HTa-based chromatin architecture to that of three histone-encoding archaea, Methanothermus fervidus, Haloferax volcanii, and Thermococcus kodakkarensis, we present evidence that HTa is an archaeal histone analog. HTa-protected fragments are GC-rich, display histone-like mono- and dinucleotide patterns around the dyad, exhibit relatively invariant positioning throughout the growth cycle, and show archaeal histone-like oligomerization dynamics. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

2019-03-11 | GSE127727 | GEO

The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog (RNA-Seq)

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Evolutionary insights into Trm112-methytransferase holoenzymes involved in translation between archaea and eukaryotes

Project description:To clarify the function of Trm112 in archaea, we have characterized structurally and functionally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies.

2018-10-22 | PXD008466 | Pride

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

Project description:Oxidative stress responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volcanii, but targets and mechanisms of actions have not been elucidated. While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the mechanisms regulating this remarkable response is lacking. Here, using a combination of high throughput and reverse molecular genetic approaches we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, aptly named Small RNA in Haloferax Oxidative Stress (SHOxi). We demonstrated that SHOxi is a functional non-coding RNA that plays gene regulatory roles in the oxidative stress response of an extremophilic archaeon. We found that SHOxi likely regulates redox homeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. The decrease in the NAD+/NADH ratio resulting from the direct RNA-RNA interaction between SHOxi and its trans-target is instrumental in the survival of H. volcanii. The regulatory effects of SHOxi provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

2020-12-08 | GSE158891 | GEO

Chromosomal origin utilisation in Haloferax meiditerranei wild type and mutant strains

Project description:We have comprehensively explored the origin utilisation in Haloferax mediterranei. Here we report three active chromosomal origins by genome-wide replication profiling, and demonstrate that when these three origins are deleted, a dormant origin becomes activated. Genomic DNA was extracted from the H. mediterranei cultures at specific time points(12h,18h,24h,36h,42h and stationary phase 66h), or during the exponential phase (OD600≈0.5) and stationary phase (OD600≈4.0)for the origin deletion strains. After being lablled, Genomic DNA from exponential phase and stationary phase were hybridized to Haloferax mediterranei genome array genechips.

2015-07-08 | E-GEOD-70597 | biostudies-arrayexpress

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