This loved ones involves E. coli RNAseH I and II , DNA transposases including the Tn5 transposase , retroviral integrases like the HIV integrase , the RuvC Holliday junction resolvase , the Argonaute RNAse , and human RNAseH 1 and 2 . The canonical RNAseH framework has about 100 aa which includes 4 conserved carboxylates that coordinate two divalent cations . The RNAseH mechanism is believed to involve each divalent cations , though a a single ion mechanism has also been proposed . The HBV RNAseH domain shares minimal but recognizable sequence identity with the RNAseH domains of reverse transcriptases as well as other retro elements . Manually optimizing alignment from the HBV RNAseH and also the HIV one RNAseH yielded 23 identity and 33 similarity . A very similar alignment concerning the HBV RNAseH along with the HIV integrase unveiled 19 identity and 33 similarity.
The HBV RNAseH is encoded with the carboxy terminus of the viral polymerase protein that also encodes the viral DNA polymerase activity . The high hydrophobicity of the HBV polymerase and its existence like a complicated with host chaperones have severely TAK-733 limited research of the HBV RNAseH. Furthermore, we demonstrated the RNAseH in its native context within the polymerase protein is unable to accept exogenous heteroduplex substrates , analogous for the inability in the DNA polymerase active web site to engage exogenous primertemplates . Consequently, almost all of our constrained understanding on the RNAseH originates from mutational studies with the viral genome within the context of viral replication carried out by us and others .
These restrictions have prevented biochemical characterization on the RNAseH and blocked biochemical screens for anti HBV RNAseH drugs to date. A couple of reports of recombinant types of the hepadnaviral RNAseH exist. Wei and enzyme inhibitor co workers expressed the HBV RNAseH domain in E. coli and purified it by denaturing nickelaffinity chromatography. Following refolding, they observed an RNAse activity. Lee et al. expressed the HBV RNAseH domain in E. coli as being a dual maltose binding protein hexahistidine fusion and purified soluble protein by two stage affinity chromatography; this enzyme had RNAseH action. Choi and co staff expressed the intact duck hepatitis B virus polymerase in yeast and reported that it had a weak RNAse activity. Lastly, Potenza et al. expressed the HBV RNAseH domain like a synthetic gene in E. coli. Following purification from inclusion bodies and refolding, this enzyme had RNAse action.
Having said that, no follow up reports have appeared with any of those techniques, probably on account of the technical problems connected to the purification protocols and or contamination problems with host RNAseH or other RNAse courses.