2010
DOI: 10.1093/nar/gkq786
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Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3′-termini for initiating DNA synthesis

Abstract: We present evidence that the reverse transcriptase (RT) of human immunodeficiency virus type-1 stabilizes in vitro very short (2-nt) duplexes of 3′-overhangs of the primer strand that are annealed to complementary dinucleotides tails of DNA or RNA template strands, provided that these sequences contain at least one C or G. This RT-induced strand ‘clamping’ activity promotes RT-directed DNA synthesis. This function is achieved only when the functional template strand is adjacent to a second DNA or RNA segment, … Show more

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Cited by 25 publications
(61 citation statements)
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“…Figure 7 shows that such template-primer substrates favored template-switching to a miRNA having a complementary 3 ′ -nucleotide residue, while an equimolar mixture of template-primer substrates with four different 3 ′ overhangs enabled more uniform template switching to miRNAs with different ends. Although retroviral RTs can template-switch by adding extra 3 ′ -nucleotide residues to cDNAs, which then base pair to the new RNA template, at least two base pairs are required, one of which must be a relatively stable GC or CG pair (Oz-Gleenberg et al 2011). The novel template-switching activity of the TeI4c-MRF RT can be used for the approach shown in Figure 7 because only a single base pair of any type is sufficient to promote template switching, even at 60°C, the operational temperature of this RT.…”
Section: Next-generation Sequencing Of Human Cdna Librariesmentioning
confidence: 99%
“…Figure 7 shows that such template-primer substrates favored template-switching to a miRNA having a complementary 3 ′ -nucleotide residue, while an equimolar mixture of template-primer substrates with four different 3 ′ overhangs enabled more uniform template switching to miRNAs with different ends. Although retroviral RTs can template-switch by adding extra 3 ′ -nucleotide residues to cDNAs, which then base pair to the new RNA template, at least two base pairs are required, one of which must be a relatively stable GC or CG pair (Oz-Gleenberg et al 2011). The novel template-switching activity of the TeI4c-MRF RT can be used for the approach shown in Figure 7 because only a single base pair of any type is sufficient to promote template switching, even at 60°C, the operational temperature of this RT.…”
Section: Next-generation Sequencing Of Human Cdna Librariesmentioning
confidence: 99%
“…Horseradish peroxidaseconjugated secondary anti-rabbit IgG antibodies were purchased from Abcam for Western blot analysis. Finally, anti-digoxigenin-POD(poly) Fab fragments from Roche were used to detect the clamp product in an enzyme-linked immunosorbent assay (ELISA) (6).…”
Section: Methodsmentioning
confidence: 99%
“…3C). This new clamp function is performed, though to variable extents, by all RTs tested but not by cellular DNA polymerases, suggesting that it is potentially essential to retroviral RTN (6). Interestingly, RTs, other than those of HIV and MLV, can also form a stable clamp with even a single nucleotide complementarity between the donor and acceptor strands, and these RTs can tolerate even short 1-to 2-nt gaps between the functional template and the adjacent strand (7).…”
mentioning
confidence: 96%
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