DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.
RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H2O2 increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.
Many bacteria containClass I CRISPR-Cas systems and utilize multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements. The activities of these complexes are described in detail for Type I effector complexes, showing CRISPR-RNA (crRNA) mediated DNA recognition that (i) relies on the presence of a short DNA sequence termed protospacer adjacent motif (PAM) and (ii) results in the recruitment of the target DNA nuclease Cas3. Type IV CRISPR-Cas systems also belong to Class I, but their substrate requirements have not been defined and a DNA nuclease has not been identified. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in PAM-dependent manner and elicits interference in the absence of DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. The deletion of the native Type IV-A CRISPR array resulted in upregulation of pilN operon transcription. Reporter gene targeting assays with endogenous and heterologous Type IV-A CRISPR-Cas machineries verified effective CRISPR interference in the absence of DNA nuclease activity. Our results demonstrate that nuclease-free Type IV-A CRISPR-Cas systems can function in host gene regulation. The observed activity resembles CRISPR interference (CRISPRi) methodology using dCas9 or Type I effectors without Cas3. Therefore, Type IV-A CRISPR-Cas activity represents a natural CRISPRi system that is found in many Pseudomonas and Klebsiella species and allows for their manipulation using synthetic crRNAs.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPRassociated (Cas) proteins elicit adaptive immunity in many bacterial species 1,2 . Foreign DNA fragments, termed protospacers, with short protospacer adjacent motifs (PAM) are captured by Cas1-Cas2 adaptation complexes and inserted into an extending CRISPR locus [3][4][5] .Transcription and processing of the CRISPR array results in mature CRISPR RNAs (crRNAs) 6,7 that guide CRISPR ribonucleoprotein complexes (crRNPs) towards complementary nucleic acid target sequences 3,[8][9][10] . Two Classes and six Types of CRISPR-Cas systems have been classified 11 and Type IV CRISPR-Cas remains as the only Type without description of its endogenous activity. Subtype IV-A CRISPR-Cas was first discovered in the genome of Acidithiobacillus ferrooxidans 12 and later shown to contain the signature proteins Csf1 and the crRNP backbone subunits Csf2 13 . Type IV-A systems are usually present on large plasmids (>200 kb) 14 and lack adaptation modules and apparent DNA nucleases like Cas3 or Cas10 13,15 . Identification of plasmid-borne protospacers targeting conjugative elements suggested that Type IV systems are involved in inter-plasmid competition 13 . The characterization of recombinant Cas proteins of the Type IV-A system of Aromatoleum aromaticum 16 indicated the presence of crRNPs that resemble Type I effectors 16,17 .Heterologous anti-plasmid interfere...
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