Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.

Sensitive detection of p53 DNA based on spatially confined fluorescence resonance energy transfer and multivalent assembly of branched DNA

We present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output, consisting of multivalent FRET pairs labeled with FAM or TAMRA.

miRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures

Reduced expression of SUMF1 was evidenced in MCF-7 cells transfected with antimiR-bDNA. Expression of miRNA-106 and 148 have positive correlation with the expression of SUMF1. miRNA-106 and 148 blocks the repressor protein controls SUMF-1 expression.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5ʹ→3ʹ (T5 Exo) and 3ʹ→5ʹ direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.