DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

The gene encoding DNA polymerase θ (Polθ) was discovered over ten years ago as having a role in suppressing genome instability in mammalian cells. Studies have now clearly documented an essential function for this unique A-family polymerase in the double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ), in metazoans. Biochemical and cellular studies show that Polθ exhibits a unique ability to perform alt-EJ and during this process the polymerase generates insertion mutations due to its robust terminal transferase activity which involves template-dependent and independent modes of DNA synthesis. Intriguingly, the POLQ gene also encodes for a conserved superfamily 2 Hel308-type ATP-dependent helicase domain which likely assists in alt-EJ and was reported to suppress homologous recombination (HR) via its anti-recombinase activity. Here, we review our current knowledge of Polθ-mediated end-joining, the specific activities of the polymerase and helicase domains, and put into perspective how this multifunctional enzyme promotes alt-EJ repair of DSBs formed during S and G2 cell cycle phases.

Key Points: Human IRAK-4 and MyD88 deficiencies impair T-independent IgM production, including IgM recognizing bacterial antigens. T-independent IgM impairment by IRAK-4 and MyD88 deficiencies is linked to inadequacy of the IgM+IgD+CD27+ B-cell subset.

The phenomenon of sexual conflict has been well documented, and in populations with biased operational sex ratios the consequences for the rarer sex can be severe. Females are typically a limited resource and males often evolve aggressive mating behaviors, which can improve individual fitness for the male while negatively impacting female condition and fitness. In response, females can adjust their behavior to minimize exposure to aggressive mating tactics or minimize the costs of mating harassment. While male-male competition is common in amphibian mating systems, little is known about the consequences or responses of females. The red-spotted newt (Notophthalmus viridescens) is a common pond-breeding amphibian with a complex, well-studied mating system where males aggressively court females. Breeding populations across much of its range have male-biased sex ratios and we predicted that female newts would have behavioral mechanisms to mitigate mating pressure from males. We conducted four experiments examining the costs and behavioral responses of female N. viridescens exposed to a male-biased environment.

Background STAT1 and STAT3 are important signaling molecules in disorders of systemic inflammation and are likely to be involved in laminitis, as laminar and systemic inflammation have been well documented in experimental models of laminitis. Hypothesis The STAT1 and STAT3 activation (via phosphorylation of tyrosine and serine moieties) is occurring in the laminar tissue during the developmental and onset of lameness time points in both the black walnut extract (BWE) and carbohydrate overload (CHO) models of laminitis. Animals Archived laminar tissue from horses. Methods Experimental studies of induced laminitis (BWE and CHO administration) in horses were conducted and laminar tissue samples archived. Western hybridization was performed to determine concentrations of Tyr- and Ser-phosphorylated STAT1 and STAT3 from these archived samples. The RT-qPCR was also performed to assess mRNA concentrations of target genes of STAT1 and STAT3. Results Increases (P < .05) in phosphorylation of tyrosine705 and serine727 of STAT3, demonstrated by band intensity ratios, are present in laminar tissue from both the BWE and CHO models at the DEV and OG1 time points. No change in phosphorylation of tyrosine701 or serine727 of STAT1 was present in the laminar tissue from either the BWE or the CHO models. The SOCS3 mRNA concentrations were increased at the onset of lameness in both the CHO and BWE models. Conclusions and Clinical Relevance The STAT3 activation likely plays a role in equine laminitis, similar to its reported involvement in organ injury/failure in human sepsis. Regulation of JAK-STAT, through STAT3 inhibitors, might serve as potential therapeutic target for controlling the inflammatory response in the septic horse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.