Initiation of DNA replication in Bacillus subtilis
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Chemistry
Abstract
Reorganising the architecture of the bacterial genetic material is a complicated and dynamic process with crucial consequences to bacterial physiology. Significant reorganisation of the genetic material is required to set the scene for the start of DNA replication and also to assist the separation of the new genetic material from the old one. The molecular mechanisms behind DNA reorganisation and remodelling are not understood. We have recently discovered that two essential replication proteins known as DnaD and DnaB from the bacterium Bacillus subtilis exhibit significant opposing DNA-remodelling activities. In an effort to establish whether these remodelling activities are of direct relevance to DNA replication and to bacterial physiology we are now concentrating our efforts to reveal the molecular mechanisms that underpin these remodelling activities and also to study the effects of limiting these two proteins in the cell. We are aiming to solve the crystal structures of the two domains of DnaD and also of the full-length protein. Mutagenesis methods in parallel with the structural studies but also assisted by the forth-coming structures, together with supercoiling and genetic complementation experiments will be used to study the mechanisms of DNA remodelling and their significance to bacterial physiology. The findings of this study will be crucial if we are to undertand how the genetic material is reorganised in relation to DNA replication in a large class of bacteria known as gram positives. This class of bacteria contains important pathogens like Staphylococcus aureus (responsible for MRSA infections) and our findings will be of general applicability to the discovery of new antibiotic targets.
Technical Summary
Remodelling of the bacterial nucleoid is a dynamic process closely associated with DNA replication. This remodelling mechanism and its close association with replication is poorly understood. With our recent work we have discovered substantial remodelling activities for two essential primosomal/replisomal proteins from Bacillus subtilis known as DnaD and DnaB. We are now seeking funding to continue our research efforts and elucidate the mechanism(s) and role(s) of these activities on the physiology of gram positive bacteria. We have designed a multi-disciplinary and complementary set of experiments, encompassing X-ray crystallography, gene complementation, atomic force microscopy and traditional mutagenesis and supercoiling experiments to achieve the two main aims described above; (i) to reveal the molecular details of the DnaD-mediated remodelling mechanism and (ii) to reveal the in vivo role of DnaD and/or DnaB on bacterial physiology. We have obtain considerable preliminary data in the form of diffracting crystals for the Nd and Cd of DnaD, topoI relaxation assays that reveal an untwisting activity for DnaD, a technical breakthrough with the isolation of whole nucleoids and are therefore in a strong position to deliver our aims and to contribute towards the understanding of an essential bacterial function for which we know precious little.
Publications
Afonso J
(2013)
Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase
in Nucleic Acids Research
Briggs GS
(2012)
Chromosomal replication initiation machinery of low-G+C-content Firmicutes.
in Journal of bacteriology
Chintakayala K
(2009)
Allosteric regulation of the primase (DnaG) activity by the clamp-loader (t) in vitro
in Molecular Microbiology
Chintakayala K
(2008)
Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome.
in Molecular microbiology
Chintakayala K
(2007)
Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues.
in Molecular microbiology
Collier C
(2012)
Untwisting of the DNA helix stimulates the endonuclease activity of Bacillus subtilis Nth at AP sites.
in Nucleic acids research
Grainger W
(2010)
DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis
in Nucleic Acids Research
Green M
(2014)
Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.
in Biosensors & bioelectronics
Larson MA
(2010)
Class-specific restrictions define primase interactions with DNA template and replicative helicase.
in Nucleic acids research
Machón C
(2010)
RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD.
in Nucleic acids research
Description | 1. Crystal structure of the N-domain of Bacillus subtilis DnaD solved. 2. The NMR structure of the C-terminal domain of DnaD was determined and the molecular details that underpin its interaction with DNA revealed. 3. The surprising structural/domain similarity of DnaD and DnaB was discovered. 4. The DNA untwisting and looping activities of DnaD were confirmed by single molecule atomic force spectroscopy. We revealed that the scaffold forming activity is required for efficient untwisting of the double helix. 5. The functions of the DnaB domains were revealed. The importance of the C-terminal domain of DnaB in oriC interaction and oligomerization were discovered, using in vivo ChIP-chip assays. 6. Using RT-PCR we revealed the expression profiles of the dnaD, dnaB and dnaI genes during the different growth phases. 7. Using DnaD and DnaB as probes in vivo we revealed that co-directional replication-transcription collisions under high growth conditions pose a significant problem in B. subtilis that requires the intervention of the replication restart machinery. All of our objectives were achieved (see above). The only objective that was not achieved was to reveal the structure of the full length DnaD protein. Instead, we have solved the structures of its individual domains; crystal structure of the scaffold forming N-terminal domain and NMR structure of the DNA-binding C-terminal domain. |
Exploitation Route | The structural information we have provided (the crystal structure of the N-terminal domain and the NMR structure of the C-terminal domain) for the essential replication initiation protein DnaD can be utilized for targeted antibiotic development against low G+C content firmicutes such as bacilli, staphylococci, streptococci and clostridia. This is a protein unique to bacteria and essential for DNA replication. |
Sectors | Chemicals Healthcare Pharmaceuticals and Medical Biotechnology |
Description | I'm a Scientist is a science dialogue event where school students talk to real scientists online for two weeks. It's in the form of an X Factor style competition between scientists, who compete for a prize of £500. For two weeks students read about the scientists' work, ask them questions, and engage in live text chats with them. The students vote for the scientist they want to get the money. The scientists with the fewest votes are evicted until only one is left to be crowned the winner. The event is supported by carefully developed and tested resources which develop students' skills and deepen their understanding. The event is funded by the Wellcome Trust. It's a fantastic science enrichment event for teaching How Science Works in schools, but more than that, it truly engages young people with real live science. Every single scientist and teacher who has taken part in the event says they would recommend it to a colleague. The event is supported by the National Science Learning Centres and by the National Coordinating Centre for Public Engagement. Science communication to School and College pupils. Advancing public understanding of Science. |
First Year Of Impact | 2009 |
Sector | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal Economic |
Title | 2V79: CRYSTAL STRUCTURE OF THE N-TERMINAL DOMAIN OF DNAD FROM BACILLUS SUBTILIS. |
Description | Crystal structure coordinates deposited in the protein data bank crystal structure coordinates for the N-terminal domain of DnaD deposited in the protein data bank (pdb; 2V79) Accession Number: DRCI:DATA2012139001760263 DOI: http://dx.doi.org/10.2210/pdb2v79/pdb crystal structure coordinates for the N-terminal domain of DnaD deposited in the protein data bank (pdb; 2V79) Accession Number: DRCI:DATA2012139001760263 DOI: http://dx.doi.org/10.2210/pdb2v79/pdb |
Type Of Material | Database/Collection of data |
Year Produced | 2008 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://dx.doi.org/10.2210/pdb2v79/pdb |
Title | GSM632110: DnaB Chip-chip in B. Subtilis grown in LB_1. |
Description | Genomic DNA from Bacillus subtilis strain 168 IPd with anti-DnaB antisera. Genomic DNA from Bacillus subtilis strain 168 IPd with anti-DnaB antisera. 1% v/v formaldehyde was added for 20 min to cross-link protein-DNA complexes. Extracted molecule genomic DNA 200-400ng ChIP or genomic DNA was labelled with Cy3 or Cy5 ChIP-chip data deposited in a public database |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
Title | GSM632111: DnaD Chip-chip in B. Subtilis grown in LB_2. |
Description | ChIP-chip data set |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
Title | GSM632112: DnaB Chip-chip in the JMA201 B. Subtilis strain in LB. |
Description | ChIP-chip data set deposited in a public microarray databse ChIP-chip datae set deposited in a public microarray database http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM632112 Genomic DNA from Bacillus subtilis strain JMA201 IPd with anti-DnaB antisera. ChIP-chip datae set deposited in a public microarray database http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM632112 |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http:///www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM632112 |
Title | GSM632114: DnaB Chip-chip in B. Subtilis grown in MM_1 |
Description | Genomic DNA from Bacillus subtilis strain 168 IPd with anti-DnaB antisera. 1% v/v formaldehyde was added for 20 min to cross-link protein-DNA complexes. An overnight culture of Bacillus subtilis (strain 168) was used to inoculate 800 ml of LeMaster minimal medium. The culture was incubated at 30°C. During exponential growth (OD595 = 0.8). 200-400ng ChIP or genomic DNA was labelled with Cy3 or Cy5 ChIP-chip data deposited in a public database |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
Description | International Scientific Interchange Scheme |
Organisation | Massachusetts Institute of Technology |
Country | United States |
Sector | Academic/University |
PI Contribution | The scheme aims to help scientists add an international dimension to their BBSRC funded research by making and establishing new contacts with international counterparts. I was awarded a grant under this scheme and also under the travelling scheme of the Royal Society to spend 4 months in MIT (School of Biology), Boston, USA as a visiting research Professor in Alan Grossman's lab. I undertook this placement between Nov 2010-Mar 2011. |
Start Year | 2010 |