at Google ScholarTitle data
Wöhrl, Birgitta M. ; Volkmann, Silke ; Moelling, Karin:
Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities.
In: Journal of Molecular Biology.
Vol. 220
(1991)
Issue 3
.
- pp. 801-818.
ISSN 0022-2836
DOI: https://doi.org/10.1016/0022-2836(91)90119-Q
Abstract in another language
The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
Further data
| Item Type: | Article in a journal |
|---|---|
| Refereed: | Yes |
| Institutions of the University: | Faculties > Faculty of Biology, Chemistry and Earth Sciences Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Apl. Prof. Dr. Birgitta Wöhrl Faculties Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors |
| Result of work at the UBT: | No |
| DDC Subjects: | 500 Science > 540 Chemistry 500 Science > 570 Life sciences, biology |
| Date Deposited: | 20 May 2019 12:15 |
| Last Modified: | 20 May 2019 12:15 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/49010 |