BIOMAT Database Logo BIOMAT Database Logo

Altering the RNase H primer grip of human immunodeficiency virus reverse transcriptase modifies cleavage specificity. 2002

Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003850 Deoxyribonuclease I An enzyme capable of hydrolyzing highly polymerized DNA by splitting phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide. This catalyzes endonucleolytic cleavage of DNA yielding 5'-phosphodi- and oligonucleotide end-products. The enzyme has a preference for double-stranded DNA. DNase I,Streptodornase,DNA Endonuclease,DNA Nicking Enzyme,DNAase I,Dornavac,Endonuclease I,Nickase,Pancreatic DNase,T4-Endonuclease II,T7-Endonuclease I,Thymonuclease,DNase, Pancreatic,Endonuclease, DNA,T4 Endonuclease II,T7 Endonuclease I
D004261 DNA Replication The process by which a DNA molecule is duplicated. Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D014158 Transcription, Genetic The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION. Genetic Transcription
D015497 HIV-1 The type species of LENTIVIRUS and the etiologic agent of AIDS. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte. Human immunodeficiency virus 1,HIV-I,Human Immunodeficiency Virus Type 1,Immunodeficiency Virus Type 1, Human
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016914 Ribonuclease H A ribonuclease that specifically cleaves the RNA moiety of RNA:DNA hybrids. It has been isolated from a wide variety of prokaryotic and eukaryotic organisms as well as RETROVIRUSES. Endoribonuclease H,RNase H,Ribonuclease H, Calf Thymus,RNAase H

Related Publications

Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Determinants of the RNase H cleavage specificity of human immunodeficiency virus reverse transcriptase.
September 1993, Nucleic acids research,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Human immunodeficiency virus reverse transcriptase-associated RNase H activity.
April 1989, The Journal of biological chemistry,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision.
July 1991, Biochemistry,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Nevirapine alters the cleavage specificity of ribonuclease H of human immunodeficiency virus 1 reverse transcriptase.
March 1995, The Journal of biological chemistry,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Inhibition of foamy virus reverse transcriptase by human immunodeficiency virus type 1 RNase H inhibitors.
July 2014, Antimicrobial agents and chemotherapy,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Template-primer binding affinity and RNase H cleavage specificity contribute to the strand transfer efficiency of HIV-1 reverse transcriptase.
August 2018, The Journal of biological chemistry,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
RNase H activity associated with reverse transcriptase from feline immunodeficiency virus.
February 1992, Journal of virology,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3).
July 1992, The Journal of biological chemistry,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity.
January 1991, The Journal of general virology,
Jason W Rausch, and Daniela Lener, and Jennifer T Miller, and John G Julias, and Stephen H Hughes, and Stuart F J Le Grice
Mutations within the primer grip region of HIV-1 reverse transcriptase result in loss of RNase H function.
April 1997, The Journal of biological chemistry,
Copied contents to your clipboard!