Faculty Opinions recommendation of DciA is an ancestral replicative helicase operator essential for bacterial replication initiation.

ABSTRACTThe accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaA’s ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

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The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00958-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 5110-5117 ◽

Cited By ~ 23

Author(s):

Carla Y. Bonilla ◽

Alan D. Grossman

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Dna Binding ◽

Replication Initiation ◽

High Rate ◽

Nucleotide Exchange ◽

Replicative Helicase ◽

Content Type ◽

Aaa Atpase ◽

Replication Initiator

ABSTRACTDnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites.Bacillus subtilisDnaD is needed during replication initiation for assembly of the replicative helicase atoriCand during replication restart at stalled replication forks. DnaD associates with DnaA atoriCand at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulateB. subtilisDnaA also affect DNA binding, whereas much of the regulation ofEscherichia coliDnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange forB. subtilisDnaA was high and not affected by DnaD. The rapid exchange is similar to that ofStaphylococcus aureusDnaA and in contrast to the low exchange rate ofEscherichia coliDnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.

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Structural insights into DNA replication initiation in Helicobacter pylori

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083673 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1632-C1632

Author(s):

Alexandre Bazin ◽

Mickaël Cherrier ◽

Laurent Terradot

Keyword(s):

Helicobacter Pylori ◽

Dna Replication ◽

Bacterial Genome ◽

Coli Strain ◽

Replication Initiation ◽

Replicative Helicase ◽

Dna Replication Initiation ◽

Terminal Domain ◽

Helicase Dnab ◽

H Pylori

In Gram-negative bacteria, opening of DNA double strand during replication is performed by the replicative helicase DnaB. This protein allows for replication fork elongation by unwinding DNA and interacting with DnaG primase. DnaB is composed of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a flexible linker. The protein forms two-tiered hexamers composed of a NTD-ring and a CTD-ring. In Escherichia coli, the initiator protein DnaA binds to the origin of replication oriC and induces the opening of a AT-rich region. The replicative helicase DnaB is then loaded onto single stranded DNA by interacting with DnaA and with the AAA+ helicase loader DnaC. However, AAA+ loaders are absent in 80% of the bacterial genome, raising the question of how helicases are loaded in these bacteria [1]. In the genome of human pathogen Helicobacter pylori, no AAA+ loader has been identified. Moreover H. pylori DnaB (HpDnaB) has the ability to support replication of an otherwise unviable E. coli strain that bears a defective copy of DnaC by complementation [2]. In order to better understand the properties of HpDnaB we have first shown that HpDnaB forms double hexamers by negative stain electron microscopy [3]. Then, we have then solved the crystal structure of HpDnaB at a resolution of 6.7Å by X-ray crystallography with Rfree/Rfactor of 0.29/0.25. The structure reveals that the protein adopts a new dodecameric arrangement generated by crystallographic three fold symmetry. When compared to hexameric DnaBs, the hexamer of HpDnaB displays an original combination of NTD-ring and CTD-ring symmetries, intermediate between apo and ADP-bound structure. Biochemistry studies of HpDnaB interaction with HpDnaG-CTD and ssDNA provides mechanistic insights into the initial steps of DNA replication in H. pylori. Our results offer an alternative solution of helicase loading and DNA replication initiation in H. pylori and possibly other bacteria that do not employ helicase loaders.

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MCM2 promotes symmetric inheritance of modified histones during DNA replication

Science ◽

10.1126/science.aau0294 ◽

2018 ◽

Vol 361 (6409) ◽

pp. 1389-1392 ◽

Cited By ~ 72

Author(s):

Nataliya Petryk ◽

Maria Dalby ◽

Alice Wenger ◽

Caroline B. Stromme ◽

Anne Strandsby ◽

...

Keyword(s):

Posttranslational Modifications ◽

Embryonic Stem ◽

Replication Initiation ◽

Genome Replication ◽

Replication Machinery ◽

Replicative Helicase ◽

Sister Chromatids ◽

Topologically Associating Domain ◽

Dna Strands ◽

Leading Strand

During genome replication, parental histones are recycled to newly replicated DNA with their posttranslational modifications (PTMs). Whether sister chromatids inherit modified histones evenly remains unknown. We measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading-strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones are inherited to sister chromatids and identifies a mechanism by which the replication machinery ensures symmetric cell division.

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Multiple mechanisms for overcoming lethal over-initiation of DNA replication

10.1101/2021.05.06.442943 ◽

2021 ◽

Author(s):

Mary E Anderson ◽

Janet L Smith ◽

Alan D Grossman

Keyword(s):

Dna Replication ◽

Double Mutant ◽

Growth Conditions ◽

Replication Initiation ◽

Cooperative Binding ◽

Positive Bacterium ◽

Replicative Helicase ◽

Synthetic Lethal ◽

Initiation Of Dna Replication ◽

Replication Initiator

DNA replication is a highly regulated process that is primarily controlled at the step of initiation. In the gram-positive bacterium Bacillus subtilis the replication initiator DnaA, is regulated by YabA, which inhibits cooperative binding at the origin. Mutants lacking YabA have increased and asynchronous initiation. We found that under conditions of rapid growth, the dnaA1 mutation that causes replication over-initiation, was synthetic lethal with a deletion of yabA. We isolated several classes of suppressors of the lethal phenotype of the ΔyabA dnaA1 double mutant. Some suppressors (dnaC, cshA) caused a decrease in replication initiation. Others (relA, nrdR) stimulate replication elongation. One class of suppressors decreased levels of the replicative helicase, DnaC, thereby limiting replication initiation. We found that decreased levels of helicase were sufficient to decrease replication initiation under fast growth conditions. Our results highlight the multiple mechanisms cells use to regulate DNA replication.

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Architecture of bacterial replication initiation complexes: orisomes from four unrelated bacteria

Biochemical Journal ◽

10.1042/bj20050143 ◽

2005 ◽

Vol 389 (2) ◽

pp. 471-481 ◽

Cited By ~ 45

Author(s):

Anna Zawilak-PAWLIK ◽

Agnieszka Kois ◽

Jerzy Majka ◽

Dagmara Jakimowicz ◽

Aleksandra Smulczyk-Krawczyszyn ◽

...

Keyword(s):

Protein Interactions ◽

Specific Binding ◽

Streptomyces Coelicolor ◽

Spatial Arrangement ◽

Replication Initiation ◽

Gradual Transition ◽

Protein Protein Interactions ◽

Bacterial Replication ◽

H Pylori ◽

Gram Negative Organisms

Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA–origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein–protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.

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