2004
DOI: 10.1038/nsmb774
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Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA

Abstract: Helicases are molecular motors that unwind double-stranded DNA or RNA. In addition to unwinding nucleic acids, an important function of these enzymes seems to be the disruption of protein-nucleic acid interactions. Bacteriophage T4 Dda helicase can displace proteins bound to DNA, including streptavidin bound to biotinylated oligonucleotides. We investigated the mechanism of streptavidin displacement by varying the length of the oligonucleotide substrate. We found that a monomeric form of Dda catalyzed streptav… Show more

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Cited by 123 publications
(157 citation statements)
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“…The Hill coefficient for unwinding from our single-molecule analysis suggests that this multimer is four or more monomers; however, because the unwinding rates are so broadly distributed in a Gaussian manner at any one RecQ concentration, this assembly can clearly be smaller (e.g., a monomer) or larger. For both Dda and NS3h, as for RecQ, the observed rate of DNA unwinding increases progressively as more monomers are accommodated in the assembly at the fork, up to a limiting value for each helicase (50,51).…”
Section: Discussionmentioning
confidence: 99%
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“…The Hill coefficient for unwinding from our single-molecule analysis suggests that this multimer is four or more monomers; however, because the unwinding rates are so broadly distributed in a Gaussian manner at any one RecQ concentration, this assembly can clearly be smaller (e.g., a monomer) or larger. For both Dda and NS3h, as for RecQ, the observed rate of DNA unwinding increases progressively as more monomers are accommodated in the assembly at the fork, up to a limiting value for each helicase (50,51).…”
Section: Discussionmentioning
confidence: 99%
“…For these two helicases, the oligomeric species is not a stable entity (52,53). In fact, for Dda, the observed Hill parameter, n, for DNA unwinding is ∼6, and biochemical analyses suggest that number of "cooperating monomers" can be as large as approximately seven to eight monomers (50). The Hill coefficient for unwinding from our single-molecule analysis suggests that this multimer is four or more monomers; however, because the unwinding rates are so broadly distributed in a Gaussian manner at any one RecQ concentration, this assembly can clearly be smaller (e.g., a monomer) or larger.…”
Section: Discussionmentioning
confidence: 99%
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“…Second, the tandem motor model predicts that translocation is unidirectional, albeit without prejudice as to which direction. To address these issues, we employed the streptavidin displacement assay developed by Kevin Raney and colleagues (35)(36)(37). 5Ј 32 P-labeled 34-mer DNA oligonucleotides containing a single biotin moiety at the fourth internucleotide from the 5Ј-end or the second internucleotide from the 3Ј-end were preincubated with excess streptavidin (SA) to form a stable SA⅐DNA complex that was easily resolved from the free biotinylated 34-mer DNA during native PAGE (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…RecQ is able to unwind dsDNA as a monomer (15,16), but on substrates with an ssDNA tail it also exhibits functional cooperativity (17) and nearly stoichiometric amounts of RecQ are required for maximal unwinding (11). Evidence for functional cooperativity from ensemble experiments has been presented for bacteriophage T4 Dda (18,19) and hepatitis C virus NS3h (20) in addition to RecQ.…”
mentioning
confidence: 94%