Biochemical characterization of a unique DNA polymerase A from the extreme radioresistant organism Deinococcus radiodurans.

Deinococcus radiodurans survives extraordinary doses of ionizing radiation and desiccation that cause numerous DNA strand breaks. D. radiodurans DNA polymerase A (DrPolA) is essential for reassembling the shattered genome, while its biochemical property has not been fully demonstrated. In this study, we systematically examined the enzymatic activities of DrPolA and characterized its unique features. DrPolA contains an N-terminal nuclease domain (DrPolA-NTD) and a C-terminal Klenow fragment (KlenDr). Compared with the Klenow fragment of E. coli Pol I, KlenDr shows higher fidelity despite the lacking of 3'-5' exonuclease proofreading activity and prefers double-strand DNA rather than Primer-Template substrates. Apart from the well-annotated 5'-3' exonuclease and flap endonuclease activities, DrPolA-NTD displays approximately 140-fold higher gap endonuclease activity than its homolog in E. coli and Human FEN1. Its 5'-3' exonuclease activity on ssDNA, gap endonuclease, and Holliday junction cleavage activities are greatly enhanced by Mn2+. The DrPolA-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation. Collectively, our results reveal distinct biochemical characteristics of DrPolA during DNA degradation and re-synthesis, which provide new insight into the outstanding DNA repair capacity of D. radiodurans.

Neurod1 modulates opioid antinociceptive tolerance via two distinct mechanisms.

BACKGROUND: The activity of neurogenic differentiation 1 (Neurod1) decreases after morphine administration, which leads to impairments of the stability of dendritic spines in primary hippocampal neurons, adult neurogenesis in mouse hippocampi, and drug-associated contextual memory. The current study examined whether Neurod1 could affect the development of opioid tolerance. METHODS: Lentivirus encoding Neurod1, microRNA-190 (miR-190), or short hairpin RNA against Neurod1 was injected into mouse hippocampi separately or combined (more than eight mice for each treatment) to modulate NeuroD1 activity. The antinociceptive median effective dose values of morphine and fentanyl were determined with tail-flick assay and used to calculate development of tolerance. Contextual learning and memory were assayed using the Morris water maze. RESULTS: Decrease in NeuroD1 activity increased the initial antinociceptive median effective dose values of both morphine and fentanyl, which was reversed by restoring NeuroD1 activity. In contrast, decrease in NeuroD1 activity inhibited development of tolerance in a time-dependent manner, paralleling its effects on the acquisition and extinction of contextual memory. In addition, only development of tolerance, but not antinociceptive median effective dose values, was modulated by the expression of miR-190 and Neurod1 driven by Nestin promoter. CONCLUSIONS: Neurod1 regulates the developments of opioid tolerance via a time-dependent pathway through contextual learning and a short-response pathway through antinociception.