5′‐Derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1

Lokhova, I A; Nevinsky, G. A.; Godovikova, Tatyana S.; Иванова, Е. М.; Koshkin, A. A.; Sergeev, Dmitry S.; Frolova, Elena I.; Rudenko, N.K.; Khomov, V.V.; Kavsan, V. M.; Зарытова, В. Ф.; Lavrik, Olga I.

doi:10.1016/0014-5793(91)80371-9

Cited by 6 publications

(5 citation statements)

References 10 publications

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“…In addition, in those substrates with a chimeric strand RT preferentially binds in the heteroduplex region, as opposed to DNA-DNA or RNA-RNA regions. This is consistent with RT binding with higher affinity to RNA-DNA versus DNA-DNA substrates (28). It is still not clear what role the length of the RNA regions may play in determining binding.…”

Section: Discussionsupporting

confidence: 72%

“…Evidence conclusively shows that second strand DNA synthesis is initiated from a unique RNA primer termed the 'polypurine tract' (8)(9)(10)(11)(12)(13)(14) and perhaps from other RNAs (29). The results shown here and those ofothers (10,22,28) suggest that the use ofRNAs as primers is likely inefficient. The polypurine tract may represent a special case in which the inability of the RT to degrade this tract allows it to be used as a primer, although inefficiently in comparison with DNA primers.…”

Section: Discussionmentioning

confidence: 64%

“…Deoxynucleotide addition by RT to such a terminus would be expected to occur efficiently if the enzyme were properly oriented on the substrate. This is as opposed to a recessed RNA 3'-terminus, where the efficiency of extension may be lower (28).…”

Section: Construction Of Substrates For Testing Hiv-rt Binding Orientmentioning

confidence: 99%

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The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

DeStefano

1995

Nucl Acids Res

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The binding of HIV reverse transcriptase (RT) to heteroduplexes was examined using a substrate consisting of a 42 nt chimeric nucleic acid composed. (5'-->3') of 23 nt of RNA and 19 of DNA. This chimera was hybridized to an internal region of a relatively long complementary DNA or RNA. When the chimera was bound to DNA and conditions limiting cleavage to a single binding event between the enzyme and substrate were employed initial RNase H-directed cleavages occurred 19-21 nt from the chimera 5'-terminus. A 42 nt strand identical in sequence to the chimera and composed of only RNA was cleaved at the same locations. Reducing the length of the DNA portion of the chimera from 19 to 7 nt did not alter the cleavage positions, suggesting that cleavage was not coordinated by the DNA 3'-terminus. Under the same conditions cleavage was not detected when the chimera was bound to RNA. In contrast, addition of dNTPs to the DNA 3'-terminus of the chimera occurred only when the chimera was bound to RNA. The results support preferable binding of RT to RNA-DNA versus DNA-DNA hybrid regions and a model in which the orientation of binding to heteroduplexes is 5'-->3' (relative to the RNA strand), polymerase to RNase H active site, with sites associated with the DNA and RNA strand respectively.

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 64%

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The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

DeStefano

1995

Nucl Acids Res

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“…However, the increased affinity can not be attributed to the RA group alone, because 3'-RA-d(pT)io and d(pT)io have similar Km values, whereas the Km for RA-d(pT)8 is 7-fold greater than that of 5'-RA-d(pT)|0. This increased stabilization has also been observed for other DNA polymerases and was attributed to the attachment of different hydrophobic residues, ethidium, phenazinium, or daunomycin, to the 5'-phosphate of the primer (Lokhova et al, 1991).…”

Section: Discussionsupporting

confidence: 64%

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase

Andréola¹,

Tarragó-Litvak²,

Левина³

et al. 1993

Biochemistry

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Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Some additional groups can stabilize DNA duplexes not only in solution, but also in their complexes with enzymes. We have shown that the attachment of phenazine, ethidium, deuteroporphyrin, or daunomycin enhanced the affinity of primers for avian myeloblastosis virus (AMV) (Lokhova et al, 1991) and HIV-1 RTs (Khodyreva et al, 1993) from 10to 20-fold. At the same time, it should be noted that the features of DNA interaction with some compounds in solution cannot always be extrapolated to the interaction of nucleic acids in complexes with enzymes, because the latter cause substantial structural changes in the nucleic acid regions situated within the protein globule (for review, see Nevinsky, 1995).…”

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confidence: 99%

Interaction of Oligonucleotides Conjugated to Substituted Chromones and Coumarins with HIV-1 Reverse Transcriptase

Martyanov¹,

Zakharova²,

Sottofattori³

et al. 1999

Antisense and Nucleic Acid Drug Development

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Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5' end of various oligonucleotides (ODN). The interaction of these compounds with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives. Some compounds activated the enzyme at relatively low concentrations (0.1-0.5 microM), followed by an inhibition of the activity at higher concentrations (5-20 microM), whereas others behave just as inhibitors. Because the presence of some coumarin or chromone derivatives conjugated to ODNs enhanced the interaction with the reverse transcriptase, we analyzed the capacity of such ODN derivatives to be used as primers. The introduction of substituent I, a chromone derivative, the 2-[(3-(aminopropyl)amino]-8-isopropyl-5-methyl-4-oxo-4H-1-benzopyran-3-c arbaldehyde], and II, a coumarin derivative, the 1-(3-aminopropoxy)-2-ethyl-3H-naphto[2,1-b]pyran-3-one, into the 5' end of a noncomplementary ODN allowed these compounds to be used as primers. In the case of complementary primers, the presence of conjugated derivatives enhanced the affinity with Km values that were two to three orders of magnitude lower than that of a complementary primer of the same length. After addition of a ddT-unit to the 3'-terminal end of the ODN, some of these primers became very effective inhibitors of RT with Ki values in the nanomolar range.

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5′‐Derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1

Cited by 6 publications

References 10 publications

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase

Interaction of Oligonucleotides Conjugated to Substituted Chromones and Coumarins with HIV-1 Reverse Transcriptase

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