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 streptavidin displacement; however, the activity increased when multiple helicase molecules bound to the biotinylated oligonucleotide. The activity does not result from cooperative binding of Dda to the oligonucleotide. Rather, the increase in activity is a consequence of the directional bias in translocation of individual helicase monomers. Such a bias leads to protein-protein interactions when the lead monomer stalls owing to the presence of the streptavidin block.

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

“…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).…”

“…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.…”

“…Elongation of this array of RecQ monomers is biased for several reasons: (i) RecQ binds most tightly to structures comprising 3′ ssDNA-tailed duplexes and ssDNAforked duplexes (7,49); (ii) RecQ unwinds the forked dsDNA structure with highest activity (7,49), resulting in net advancement; and (iii) monomers behind the lead helicase can rapidly translocate independently in the 3′→ 5′ direction (21,22), because no DNA unwinding is necessary. Based on ensemble data, related models were proposed for the bacteriophage T4 Dda, hepatitis C virus NS3 helicases (NS3h) and RecQ (27,50,51). In the case of RecQ, three to six monomers could functionally interact to unwind DNA with ssDNA extensions (27).…”

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

“…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.…”

Fine tuning of a DNA fork by the RecQ helicase

“…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.…”