Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones

Hyperosmolarity Attenuates the Contraction of Rat Trachea Through the Inhibition of Phosphatidylinositol Response

Although hyperosmolarity associated with diabetes is known to attenuate contractile response of airway smooth muscle, intracellular mechanisms involved are not fully understood. We examined the effects of hyperosmolarity on carbachol (CCh)- and aluminum fluoride (AF)-induced contractile and phosphatidylinositol (PI) responses of rat trachea. In vitro measurements of isometric tension and [3H] inositol monophosphate (IP1) formed were conducted by using rat tracheal rings and slices. Hyperosmolarity solutions of 350, 450 and 600 mOsm were made with dissolving glucose in Krebs-Henseleit (K-H) solution. Hyperosmolarity attenuated dose-dependently CCh-induced contraction of rat trachea (1.86 ± 0.13 g at 300 mOsm, 1.85 ± 0.16 g at 350 mOsm, 1.37 ± 0.07 g at 450 mOsm and 0.50 ± 0.04 g at 600 mOsm, respectively), and also attenuated CCh- induced IP1 accumulation (5.77 ± 0.33 Bq at 300 mOsm, 3.38 ± 0.26 Bq at 350 mOsm, 2.08 ± 0.30 Bq at 450 mOsm and 1.71 ± 0.40 Bq at 600 mOsm, respectively), and AF-induced IP1 accumulation (3.93 ± 0.22 Bq at 300 mOsm, 1.63 ± 0.14 Bq at 450 mOsm and 1.02 ± 0.14 Bq at 600 mOsm, respectively). The results suggest that hyperosmolarity would inhibit G-protein-coupled phospholipase C, resulting in attenuation of CCh-induced airway smooth muscle contraction

UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G1 phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G2 phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G1 by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced "clean" DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced "dirty" DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ