Computational design of a thermolabile uracil-DNA glycosylase of Escherichia coli.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step.

Multiple hTAF(II)31-binding motifs in the intrinsically unfolded transcriptional activation domain of VP16.

Transcriptional activation domain (TAD) in virion protein 16 (VP16) of herpes simplex virus does not have any globular structure, yet exhibits a potent transcriptional activity. In order to probe the structural basis for the transcriptional activity of VP16 TAD, we have used NMR spectroscopy to investigate its detailed structural features. Results show that an unbound VP16 TAD is not merely "unstructured" but contains four short motifs (residues 424-433, 442-446, 465-467 and 472-479) with transient structural order.

Backbone resonance assignment of a proteolysis-resistant fragment in the oxygen-dependent degradation domain of the hypoxia inducible factor 1alpha.

Hypoxia-inducible factor 1alpha (HIF1alpha) is a transcription factor that plays a key role in the adaptation of cells to low oxygen stress and oxygen homeostasis. The oxygen-dependent degradation (ODD) domain of HIF1alpha responsible for the negative regulation of HIF1alpha in normoxia is intrinsically unfolded. Here, we carried out the backbone (1)H, (15)N, and (13)C resonance assignment of a proteolysis-resistant fragment (residues 404-477) in the HIF1alpha ODD domain using NMR spectroscopy.

Unique substrate spectrum and PCR application of Nanoarchaeum equitans family B DNA polymerase.

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N.

Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1.

Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized.

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans.

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes.