Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB

Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle

Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo

Hepatocellular carcinoma (HCC), a highly aggressive liver cancer, is a leading cause of cancer-related death. Tumor Treating Fields (TTFields) are electric fields that exert antimitotic effects on cancerous cells. The aims of the current research were to test the efficacy of TTFields in HCC, explore the underlying mechanisms, and investigate the possible combination of TTFields with sorafenib, one of the few front-line treatments for patients with advanced HCC. HepG2 and Huh-7D12 human HCC cell lines were treated with TTFields at various frequencies to determine the optimal frequency eliciting maximal cell count reduction. Clonogenic, apoptotic effects, and autophagy induction were measured. The efficacy of TTFields alone and with concomitant sorafenib was tested in cell cultures and in an orthotopic N1S1 rat model. Tumor volume was examined at the beginning and following 5 days of treatment. At study cessation, tumors were weighed and examined by immunohistochemistry to assess autophagy and apoptosis. TTFields were found in vitro to exert maximal effect at 150 kHz, reducing cell count and colony formation, increasing apoptosis and autophagy, and augmenting the effects of sorafenib. In animals, TTFields concomitant with sorafenib reduced tumor weight and volume fold change, and increased cases of stable disease following treatment versus TTFields or sorafenib alone. While each treatment alone elevated levels of autophagy relative to control, TTFields concomitant with sorafenib induced a significant increase versus control in tumor ER stress and apoptosis levels, demonstrating increased stress under the multimodal treatment. Overall, TTFields treatment demonstrated efficacy and enhanced the effects of sorafenib for the treatment of HCC in vitro and in vivo, via a mechanism involving induction of autophagy

Correction: Davidi et al. Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma <i>In Vitro</i> and <i>In Vivo</i>. <i>Cancers</i> 2022, <i>14</i>, 2959

The authors wish to make minor corrections to Figure 1 and Figure 2 of the following paper [...