Biomedical subjects

R G Buiser

Publications and source records attributed to R G Buiser.

5 recordsLinked to original sources

PubMed1993

Pausing by retroviral DNA polymerases promotes strand transfer from internal regions of RNA donor templates to homopolymeric acceptor templates.

We have examined the ability of reverse transcriptases (RT) to catalyze strand transfer from internal regions of RNA templates, resulting in switching of a primer from one template to another. To study this phenomenon, we employed a system of donor and acceptor templates in which homologous strand transfer can occur from a homopolymeric sequence, positioned internally on the donor template. Our results indicate that reverse transcriptases from human immunodeficiency virus (HIV), avian myeloblastosis virus (AMV), and murine leukemia virus (MuLV) are all able to catalyze strand transfer from this sequence. Catalysis of this reaction is not dependent upon ribonuclease H (RNase H) activity, since an RNase H-deficient form of HIV-RT is able to catalyze the reaction efficiently. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity, did not inhibit the template switching by either the native or RNase H-deficient forms of HIV-RT. Our data further indicate that template switching may be promoted by RT pausing at a specific site on the donor template. Conditions that increase RT pausing at this site also increase template switching. These results suggest that transient RT pausing at specific sites on the viral genome during reverse transcription may promote template switches that in turn lead to recombination.

DNA-Directed DNA Polymerase↗

PubMed1992

Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates.

We have examined the parameters that determine the length and distribution of products synthesized processively by the human immunodeficiency virus reverse transcriptase (HIV-RT). On native or homopolymer templates, the overall length distribution of processively synthesized products is increased by increased temperature or deoxynucleoside triphosphate concentration, or decreased ionic strength. Specific terminations of processive synthesis on either native DNA or RNA templates occur most frequently at positions where the reverse transcriptase (RT) pauses during synthesis. These sites correlate with the template sequence 3'-(A/U)(A/U)(G/C)-5', particularly when this sequence is predicted to be base paired with another region of the template in a secondary structure. Many positions of termination are in similar positions on DNA or RNA templates. Notable exceptions are runs of A residues, which promote termination on DNA but not RNA templates. Termination intensities vary when different RTs are used demonstrating an influence of RT structure.

Base Sequence↗

PubMed1991

Human immunodeficiency virus reverse transcriptase displays a partially processive 3' to 5' endonuclease activity.

We have examined the ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV-RT) using a natural sequence 83-nucleotide-long RNA template to which was hybridized a DNA oligomer. This substrate configuration allowed for the simultaneous electrophoretic resolution of 5'-, 3'-, and internally derived RNase H cleavage products. Assays performed in the presence of excess challenger RNA to sequester the RT permitted the analysis of products resulting from a single round of binding of RT to substrate. Substrate cleavage was highly sensitive to ionic strength, showing greatest activity at low KCl concentrations. The increase in cleavage correlated with an increase in the half-life of the enzyme on the RNA-DNA hybrid from approximately 31 s to 6.2 min at 80 and 5 mM KCl, respectively. Internally derived cleavage products generated in challenged reactions were primarily 2-9 nucleotides in length. These lengths indicate that the products were generated by an endo- rather than an exonuclease activity. The directionality and processivity of the endonuclease were also determined by examination of cleavage products from challenged reactions. Although the lengths of 5'-derived products markedly decreased with time, no change in the size distribution of 3'-derived products was observed, indicating that cleavage proceeded processively in the 3' to 5' direction. The 5'-derived products were shortened more in reactions performed under conditions allowing multiple versus single enzyme-binding events, suggesting that the endonuclease action of a single enzyme is not processive enough to generate the maximum possible amount of cleavage on each substrate. Therefore, HIV-RT displays a partially processive 3' to 5' endonuclease activity.

DNA↗

PubMed1991

Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases.

We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA). In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made. Models of retrovirus replication require the RT to catalyze two distinct strand transfers. Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers. RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition. However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products. An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis. Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction. RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis. However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e. conditions that simulate higher processivity, allow strand transfer synthesis. Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated. These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs.

Avian Myeloblastosis Virus↗

PubMed1991

Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled.

The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.

Avian Myeloblastosis Virus↗