Methods of analyzing processivity.
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Animals↗
Biomedical subjects
L M Mallaber
Publications and source records attributed to L M Mallaber.
Quantitative analysis of RNA cleavage during RNA-directed DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases.
We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs). Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer. The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H. RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated. Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products. Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity. Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity. The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater. They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand. In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed.
Avian Myeloblastosis Virus↗
Determinants of the RNase H cleavage specificity of human immunodeficiency virus reverse transcriptase.
We examined the ribonuclease H (RNase H) specificity of human immunodeficiency virus reverse transcriptase (HIV-RT) using heteropolymeric RNAs hybridized to complementary DNAs. Experiments were performed in the presence of excess challenger polymer (poly(rA)-oligo(dT)) to reveal cleavages resulting from single enzyme binding events. Previous results suggested that initial RNase H directed cleavages were a fixed distance from a DNA primer terminus recessed on an RNA template, i.e. determined by the binding position of the polymerase active site. The influences of recessed RNA termini were not evaluated. In current experiments, RNAs that were 30, 42, or 50 nucleotides long were hybridized to the same 88 nucleotide long complementary DNA, such that the 5' terminal nucleotide of each RNA was hybridized to the 29th nucleotide from the 3' end of the DNA. In all three cases the RNA was initially cleaved between the 19th and 21st nucleotides from its 5' end. Thus, cleavage was not coordinated by the recessed 3' terminus of the RNA. Subsequent cleavages in either direction on the RNA were also observed. An insertion within the RNA that moved the preferred initial cut sequence 10 nucleotides further from the 5' end of the RNA decreased but did not abolish cleavage at the sequence. However, changing the nucleotide sequence in the region of the preferred cleavage either by the insertion experiment or mutagenesis did not significantly alter its capacity for cleavage. These results demonstrated a dominant position preference, plus a sequence priority. In another experiment, a 25 nucleotide long DNA was hybridized such that its 3' terminal nucleotide was 9 nucleotides from the 5' end of a 60 nucleotide complementary RNA. The preferred RNA cleavage sequence discussed above, was 10-14 nucleotides upstream of the 3' end of the DNA. However, initial cleavages occurred 17-20 nucleotides from the DNA 3' end, consistent with cleavage being coordinated by the recessed 3' terminus of the DNA primer.
Base Sequence↗
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↗
Structural relationships between two forms of DNA polymerase epsilon from calf thymus.
We previously reported purification of two forms of DNA polymerase epsilon from calf thymus (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). We have now used the "polymerase trap" photolabeling method to identify the polypeptides containing the polymerase active site in each enzyme preparation. The molecular mass of these polypeptides are 210 and 145 kDa for the polymerases now designated epsilon and epsilon*, respectively. Renaturation of polymerase activity from denaturing gel electrophoresis corroborates the polymerase trap results. Photolabeling of polymerase fractions suggests that the smaller subunit is derived by proteolysis of the larger subunit during purification. Native sedimentation coefficient measurements of polymerase-containing column fractions further suggest a precursor/product relationship between the two polymerases. Response of polymerization activity to a battery of inhibitors normally used to distinguish mammalian nuclear DNA polymerases was found to be essentially identical for polymerases epsilon, epsilon*, and the epsilon* generated in fractions initially containing epsilon. These latter results demonstrate that the loss of the protease-sensitive domain of the active site subunit does not affect catalytic function as measured in a standard DNA polymerase assay. The sole apparent functional difference observed here between the epsilon and epsilon* forms is evidence that only the full-length epsilon form can be directly photocrosslinked to dATP, independent of DNA synthesis. Photolabeling of the post-microsomal supernatant fraction from thymus glands obtained from fetal calves reveals the presence of both the epsilon and epsilon* polypeptide.
Animals↗
Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase.
We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.
Base Sequence↗
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↗
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↗
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↗