Nucleic Acids Research, 1994, Vol. 22, No. 18 3793-3800
© 1994
ENZYMOLOGY |
Quantitative analysis of RNA cleavage during RNAdirected DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases
Department of Microbiology, University of Maryland College Park MD 20742 1Departments of Biochemistry, University of Rochester 601 Elmwood Avenue, Rochester, NY 1462, USA 2Departments of Medicine, University of Rochester 601 Elmwood Avenue, Rochester, NY 1462, USA 3Departments of Cancer Center, University of Rochester 601 Elmwood Avenue, Rochester, NY 1462, USA
*To whom correspondence should be addressed
Received April 4, 1994. Revised August 5, 1994. Accepted August 5, 1994.
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 HIVand 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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Song, V. P. Basu, M. N. Hanson, B. P. Roques, and R. A. Bambara Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer J. Biol. Chem., February 8, 2008; 283(6): 3141 - 3150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Kupfer, C. Crey-Desbiolles, and C. J. Leumann Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template Nucleic Acids Res., November 29, 2007; 35(20): 6846 - 6853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Held, J. D. Kissel, D. Saran, D. Michalowski, and D. H. Burke Differential Susceptibility of HIV-1 Reverse Transcriptase to Inhibition by RNA Aptamers in Enzymatic Reactions Monitoring Specific Steps during Genome Replication J. Biol. Chem., September 1, 2006; 281(35): 25712 - 25722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Schultz, M. Zhang, and J. J. Champoux Sequence, Distance, and Accessibility Are Determinants of 5'-End-directed Cleavages by Retroviral RNases H J. Biol. Chem., January 27, 2006; 281(4): 1943 - 1955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Derebail and J. J. DeStefano Mechanistic Analysis of Pause Site-dependent and -independent Recombinogenic Strand Transfer from Structurally Diverse Regions of the HIV Genome J. Biol. Chem., November 12, 2004; 279(46): 47446 - 47454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-X. Wilhelm, M. Wilhelm, and A. Gabriel Extension and Cleavage of the Polypurine Tract Plus-strand Primer by Ty1 Reverse Transcriptase J. Biol. Chem., November 28, 2003; 278(48): 47678 - 47684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Schultz, M. Zhang, and J. J. Champoux Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus J. Virol., May 1, 2003; 77(9): 5275 - 5285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wisniewski, Y. Chen, M. Balakrishnan, C. Palaniappan, B. P. Roques, P. J. Fay, and R. A. Bambara Substrate Requirements for Secondary Cleavage by HIV-1 Reverse Transcriptase RNase H J. Biol. Chem., August 2, 2002; 277(32): 28400 - 28410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. F. Gerard, R. J. Potter, M. D. Smith, K. Rosenthal, G. Dhariwal, J. Lee, and Deb. K. Chatterjee The role of template-primer in protection of reverse transcriptase from thermal inactivation Nucleic Acids Res., July 15, 2002; 30(14): 3118 - 3129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wilhelm, O. Uzun, E. H. Mules, A. Gabriel, and F.-X. Wilhelm Polypurine Tract Formation by Ty1 RNase H J. Biol. Chem., December 7, 2001; 276(50): 47695 - 47701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wisniewski, M. Balakrishnan, C. Palaniappan, P. J. Fay, and R. A. Bambara Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H PNAS, October 12, 2000; (2000) 210392297. [Abstract] [Full Text]
|
|
|
|
|
|
M. D. Driscoll and S. H. Hughes Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences J. Virol., October 1, 2000; 74(19): 8785 - 8792. [Abstract] [Full Text]
|
|
|
|
|
|
C. D. Kelleher and J. J. Champoux RNA Degradation and Primer Selection by Moloney Murine Leukemia Virus Reverse Transcriptase Contribute to the Accuracy of Plus Strand Initiation J. Biol. Chem., April 21, 2000; 275(17): 13061 - 13070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Schultz, M. Zhang, C. D. Kelleher, and J. J. Champoux Polypurine Tract Primer Generation and Utilization by Moloney Murine Leukemia Virus Reverse Transcriptase J. Biol. Chem., December 3, 1999; 274(49): 34547 - 34555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Noad, N. S. Al-Kaff, D. S. Turner, and S. N. Covey Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements J. Biol. Chem., December 4, 1998; 273(49): 32568 - 32575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gotte, G. Maier, H. J. Gross, and H. Heumann Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals J. Biol. Chem., April 24, 1998; 273(17): 10139 - 10146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Kelleher and J. J. Champoux Characterization of RNA Strand Displacement Synthesis by Moloney Murine Leukemia Virus Reverse Transcriptase J. Biol. Chem., April 17, 1998; 273(16): 9976 - 9986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Palaniappan, J. K. Kim, M. Wisniewski, P. J. Fay, and R. A. Bambara Control of Initiation of Viral Plus Strand DNA Synthesis by HIV Reverse Transcriptase J. Biol. Chem., February 13, 1998; 273(7): 3808 - 3816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Palaniappan, M. Wisniewski, P. S. Jacques, S. F.J. Le Grice, P. J. Fay, and R. A. Bambara Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function J. Biol. Chem., April 25, 1997; 272(17): 11157 - 11164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. DeStefano and J. J. DeStefano Interaction of Human Immunodeficiency Virus Nucleocapsid Protein with a Structure Mimicking a Replication Intermediate. EFFECTS ON STABILITY, REVERSE TRANSCRIPTASE BINDING, AND STRAND TRANSFER J. Biol. Chem., July 5, 1996; 271(27): 16350 - 16356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Palaniappan, G. M. Fuentes, L. Rodríguez-Rodríguez, P. J. Fay, and R. A. Bambara Helix Structure and Ends of RNA/DNA Hybrids Direct the Cleavage Specificity of HIV-1 Reverse Transcriptase RNase H J. Biol. Chem., January 26, 1996; 271(4): 2063 - 2070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuentes, L. Rodríguez-Rodríguez, P. J. Fay, and R. A. Bambara Use of an Oligoribonucleotide Containing the Polypurine Tract Sequence as a Primer by HIV Reverse Transcriptase J. Biol. Chem., November 24, 1995; 270(47): 28169 - 28176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Schultz, S. H. Whiting, and J. J. Champoux Cleavage Specificities of Moloney Murine Leukemia Virus RNase H Implicated in the Second Strand Transfer During Reverse Transcription J. Biol. Chem., October 13, 1995; 270(41): 24135 - 24145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Schultz, M. Zhang, C. D. Kelleher, and J. J. Champoux Analysis of Plus-strand Primer Selection, Removal, and Reutilization by Retroviral Reverse Transcriptases J. Biol. Chem., October 6, 2000; 275(41): 32299 - 32309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wisniewski, M. Balakrishnan, C. Palaniappan, P. J. Fay, and R. A. Bambara The Sequential Mechanism of HIV Reverse Transcriptase RNase H J. Biol. Chem., November 22, 2000; 275(48): 37664 - 37671. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wisniewski, M. Balakrishnan, C. Palaniappan, P. J. Fay, and R. A. Bambara Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H PNAS, October 24, 2000; 97(22): 11978 - 11983. [Abstract] [Full Text] [PDF]
|
|