Monthly Archives: March 2012

Double-strand break interacting RNAs (diRNAs)

A new role for small RNAs in the repair of DNA double-strand breaks has been reported in Cell. Wei et al. have found diRNAs, derived from the vicinity of DNA double-strand breaks, in both Arabidopsis thaliana and human cells.

DNA double strand breaks (DSBs) are a particularly deleterious form of DNA damage as they can cause chromosomal translocations and induce cell death. To maintain the genome’s integrity, eukaryotic cells employ two different mechanisms of DSB repair. Non-homologous end joining (NHEJ) is an efficient mechanism that rapidly repairs DSBs without requiring an homologous template. However, NHEJ often causes insertions or deletions at the break site. Homologous recombination (HR) is a less error prone mechanism in which a sister chromatid is used as a template for repair. A specialised form of HR, single-strand annealing (SSA) that doesn’t require a sister chromatid can take place at repetitive sequences.

Wei et al. used an assay system that monitors DSB repair by SSA in the model plant Arabidopsis thaliana. A genetic cross causes a single DSB in an inactive reporter gene containing a repeat. SSA mediated repair restores the activity of the reporter gene and allows a quantitative and visible readout of DSB repair events. For instance, when this assay system is introduced (by crossing) into a genetic background mutant for atr (encoding a PI3 kinase known to be involved in DSB response), the researchers observed a large reduction in repair efficiency.

The first clue that suggested that small RNAs may be involved in double strand break repair came when they crossed their DSB repair assay system into lines mutant for Dicer-like proteins (DCL). Dicer and DCLs are responsible for the biogenesis of small RNAs (miRNAs and siRNAs) from double-stranded RNAs. Mutations in three different dcl genes (especially dcl3) all diminished the efficiency of DSB repair. The researchers therefore tried to examine whether small RNAs were produced from sequences adjacent to the DSB site. By probing northern blots with sequence flanking the DSB site, Wei et al detected a population of small RNAs approximately 21nt in length that were only present when DSBs had been induced. Deep sequencing (direct sequencing of RNA populations) revealed that these DSB-induced small RNAs (diRNAs) were specifically produced from the sequences flanking the DSB (approximately 800bp in each direction) and derived from both the sense and antisense strands in equal measure. By using a similar assay that monitored DSB repair by HR, they showed that diRNAs were also produced in this system.

In plants, a well characterised small RNA system mediates heterochromatic silencing of repetitive sequences by DNA methylation. Wei et al. used this pathway as a model to dissect the diRNA system. In the heterochromatic-siRNA system, single stranded RNA transcripts generated by the DNA-dependent RNA polymerase IV (Pol IV)  are converted to dsRNAs by the action of the RNA-dependent RNA polymerase 2 (RDR2). The dsRNAs are then cleaved into hc-siRNAs by Dicer-like proteins. When complexed with the Argonaute protein AGO4, hc-siRNAs direct de novo DNA methylation. By using the DSB assay system in backgrounds mutant for these factors and deep sequencing, Wei et al.  found that diRNA production requires the activity of Pol IV, RDR2 and RDR6 and DCLs, and that this pathway is under the control of DSB responsive kinase ATR. However, diRNA-mediated DSB repair does not involve RNA-directed DNA methylation pathway effector components such as AGO4. Instead, a different Argonaute protein AGO2 was found to complex diRNAs. Both diRNA accumulation and DSB repair were compromised in ago2 mutants.

Wei et al. went on to enquire as to whether diRNAs are involved in DSB repair in animals as well as plants. Using a similar HR mediated DSB repair assay in a human cell line, they showed small RNAs are also produced close to DSBs. Interestingly, whereas in plants the diRNAs were produced from sequences immediately neighbouring the DSB, in human cells they originated from a broader vicinity around the break site and not immediately adjacent. When Dicer or Ago2 were depleted in human cells DSB repair was compromised.

This paper has demonstrated the existence of a new class of small RNAs and their involvement in yet another important biological process. However, the details of how diRNAs act in DSB repair are completely unknown as yet. The authors suggest that diRNAs may guide histone modifications around the DSB site that facilitate DNA repair. Alternatively, diRNA-AGO2 complexes may be directly target DSB repair complexes to break sites. The assay systems used in this study only tested DSB repair by HR and SSA. It would be interesting to know whether diRNAs are also involved in DSB repair by NHEJ.

Wei, W., Ba, Z., Gao, M., Wu, Y., Ma, Y., Amiard, S., White, C., Danielsen, J., Yang, Y., & Qi, Y. (2012). A Role for Small RNAs in DNA Double-Strand Break Repair Cell DOI: 10.1016/j.cell.2012.03.002

Transposons and plasmids combine for bacterial chromosomal transfer.

A mechanism that facilitates the horizontal transfer of large segments of chromosomal DNA has been discovered in natural isolates of the pathogenic bacterium Yersinia pseudotuberculosis.

Although horizontal gene transfer (HGT) is recognised as a major force in bacterial evolution, and many mobile genetic elements underlying genomic plasticity have been characterised, the mechanisms by which genetic exchange of chromosomal DNA occurs in natural populations have remained largely hypothetical.

In experiments analysing the mobility of a pathogenicity island (HPI) in Yersinia pseudotuberculosis, Lesic and Carniel (2005) discovered that HPI could be transferred between natural isolates in a process that occurred optimally at 4˚C. However, this process didn’t require integration/excision machinery encoded in the HPI, and also transferred adjacent sequences (>46kb). To test whether this process was specific to the HPI region, Lesic et al inserted various antibiotic resistance markers equidistantly within the Y. pseudotuberculosis chromosome. When this strain was co-incubated with a naïve recipient strain, transfer of these markers was observed in ‘transconjugants’. None of the transconjugants acquired more than one of the antibiotic resistance loci, showing that the transferred chromosomal fragments were less that 1.5Mb in size. The transfer mechanism was also capable of mediating the transmission of a non-mobilisable plasmid (pUC4K). Together these results showed the existence of a mechanism for generalised DNA transfer that functioned at low temperature (termed GDT4).

Lesic et al. found that the GDT4 performing strain contained a very high molecular weight plasmid (≥100Mb). When this plasmid was removed (‘cured’) from the strain, GDT4 was abolished. They therefore termed this plasmid pGDT4. pGDT4 was transferred during transconjugation experiments and was able to confer the ability to transfer chromosomal DNA.

Electron micrograph of aggregating Y. pseudotuberculosis. White arrows point to bridge-like structures.

Sequencing of pGDT4 showed that it contained genes involved in conjugation (the process by which a pilus acts as a conduit for transfer of DNA between cells). When part of this conjugative machinery was deleted GDT4 ability was lost. Interestingly though, pili could not be observed by electron microscopy, and strong shaking of cultures (predicted to disrupt pilus-mediated interactions) had no effect. Instead, the bacteria were seen to tightly aggregate, and seemed to be connected by ‘bridges’.

Organisation of pUC4K plasmids from transconjugants, showing novel IS insertions and IS mediated novel organisations.

pGDT4 also contains a number of Insertion Sequences (IS, short bacterial transposable elements). None of these IS were present on the bacterial chromosome or pUC4K (the non-mobilisable plasmid). After GDT4 transfer of pUC4K, it was observed the size of pUC4K was often altered. Of 10 transconjugant pUC4Ks measured, 3 were the original size, but (pGDT4 derived) IS insertions had lengthened the others. In 5 cases a single copy of ISYps1 had been acquired by pUC4K, whilst in 1 case a large section of pGDT4 had been inserted between two ISYps3 elements.

ISYps1 and ISYps3 (members of the IS6 and Tn3 families respectively) transpose by a mechanism termed replicative transposition. An intermediate stage during this mode of transposition is the formation of a ‘co-integrate’ in which the donor and target replicons (in this case pGDT4 and pUC4K) are fused by 2 copies of the transposon. The cointegrate is then resolved by homologous recombination, leaving a new copy of the transposon on the target and the original still in place on the donor.

Model describing ISYps1 transposition mediated mobilisation of pUC4K via cointegrate formation.

These observations suggested that GDT4 occurs by cointegrates of pGDT4 and other replicons (bacterial chromosome or other plasmids) being formed during IS transposition and then being transferred to recipient cells by conjugation. In further experiments, Lesic et al. confirmed that replicative transposition of ISYps1 was capable of  driving cointegrate formation between different plasmids in E.coli, that could also be transferred by conjugation.

This mode of chromosomal transfer is very similar to the ‘Hfr‘ system characterised in E. coli. In Hfr strains, transfer of chromosomal DNA occurs by conjugation because the conjugative plasmid ‘F’ becomes integrated into the chromosome. Integration of F occurs by homologous recombination at IS sequences shared between the plasmid and the bacterial chromosome. The difference in GDT4 is that no sequence identity between the two replicons is necessary. ISYps1 transposition does not seem to target any particular sequence. This makes this mode of chromosomal conjugative transfer less constrained than Hfr and potentially more powerful. It is notable that plasmids often carry a large number of IS. For instance, a plasmid in Shigella carries 93 copies of IS of 21 different types. The ability to confer chromosomal DNA transfer may be a selective advantage underlying plasmid/IS symbioses.

GDT4 was optimal at low temperature and low iron concentration. However, pGDT4 did not encode any known thermoregulators. This suggests that temperature sensitive pGDT4 conjugation is under the control host genes. GDT4 could therefore be a natural response to challenging growth conditions.

Lesic, B., Zouine, M., Ducos-Galand, M., Huon, C., Rosso, M., Prévost, M., Mazel, D., & Carniel, E. (2012). A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis PLoS Genetics, 8 (3) DOI: 10.1371/journal.pgen.1002529

The Toxic Nano-Syringe of Vibrio cholerae.

Type VI secretion systems are used by bacteria to inject toxins into both bacterial competitors and host cells during pathogenesis. A new paper in Nature visualises these nanomachines and shows that they act by the swift contraction of a bacteriophage tail-like tube.

Bacterial type VI secretion systems (T6SS) are used to kill both eukaryotic and prokaryotic cells through the translocation of toxic proteins in a cell-cell contact-dependent process. T6SS encoding clusters of 15-20 genes are found in pathogenicity islands in the genomes of about a quarter of all sequenced gram –ve bacteria. Many T6SS proteins show sequence or structural homology with components of contractile phage tails. For instance, VgrG and Hcp, proteins secreted by T6SS, are structural homologues of a phage needle protein and phage tail tube protein respectively. Likewise, two Vibrio cholerae T6SS components VipA and VipB form tubular structures that resemble the tail sheath of T4 bacteriophage.

To investigate the role of VipA in T6SS, Basler et al. tagged VipA with a green fluorescent protein (GFP) and visualised it’s dynamics in cells. They found that the VipA-GFP fusion proteins were associated with long straight structures in the cell. These were often as long as the width of the cell (0.75-1µm) and varied in number between 0 and 5. By using time-lapse imaging, the researchers found that these putative sheath structures underwent a dynamic pattern of assembly, contraction and disassembly. The extended sheath assembled at a rate of 20-30 s µm-1. Sheaths then contracted to about 50% of their extended length in approximately 5ms. The contracted sheath was then disassembled in 30-60s.

Electron cryotomographic slices of V. cholerae showing Type VI secretion systems in the cytosol. In the left panel the T6SS is in the extended form, in the right it is contracted.

Basler et al. went on to visualise the T6SS sheaths directly using electron cryotomography. This discerned long straight tubular structures that existed in two conformations: A longer and thinner extended structure and a shorter and wider contracted form (see figure). The tubes were connected to the inner membrane by a flared bell-shaped base. Distal to the base in the extended T6SS structures was ‘conical-shaped density’ that crossed the periplasm and protruded through the outer membrane.

The results described in this paper are consistent with a model of T6SS action in which Hcp forms an inner tube within the VipA/VipB sheath. The Hcp tube, tipped with a VgrG ‘needle’ is fired into the target cell membrane by contraction of the T6SS sheath. Thus the energy captured by conformational change of the VipA/VipB polymeric sheath transports the toxic proteins through the cell membrane.

Basler, M., Pilhofer, M., Henderson, G., Jensen, G., & Mekalanos, J. (2012). Type VI secretion requires a dynamic contractile phage tail-like structure Nature, 483 (7388), 182-186 DOI: 10.1038/nature10846