Multiple Escherichia coli RecQ Helicase Monomers Cooperate to Unwind Long DNA Substrates

Abstract

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37°C, whereas only modest or no cooperativity was observed at 25°C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37°C but not at 25°C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.Helicases are molecular motor enzymes that unwind and translocate nucleic acids in an ATP hydrolysis-dependent manner (1, 2). RecQ DNA helicases constitute a ubiquitous family of helicases that play a key role in maintaining genome stability in a wide range of organisms from bacteria to higher eukaryotes (for a review, see Ref.3). These enzymes are involved in many of the processes of DNA metabolism, including recombination, DNA replication, and DNA repair (3-5). In humans, defects in RecQ family helicases, encoded by the blm, wrn, and RecQ4 genes, give rise to the Bloom, Werner, and Rothmund-Thomson syndromes, respectively, characterized by genomic instability and susceptibility to cancer. The Escherichia coli RecQ helicase, the prototype enzyme of this family, is involved in various processes, including the homologous recombination and double strand break repair mediated by the RecF machinery (6) as well as suppression of illegitimate recombination (7).The structure-function relationship of helicases is difficult to understand at the molecular level, because proteins with different organizations may have similar activities. For example, the need for an oligomeric structure for helicase activity strongly varies between helicase families. Hexameric rings are required for active E. coli Dn...

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

“…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). Similar to RecQ, even though monomers of Dda and NS3h can unwind appropriate dsDNA substrates, linear arrays of several monomers of Dda and NS3h are inferred to cooperate to enhance the rate and effectiveness of DNA unwinding.…”

“…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).…”

“…RecQ binds DNA with Hill coefficients that vary between 1 and 2, depending on the number and length of ssDNA tails attached to the duplex DNA, indicating that a dimer is formed upon binding to DNA (27). Additionally, RecQ binds ssDNA in a cooperative fashion (ω = 25), establishing that the monomers interact with a 25-fold higher affinity to each other when bound to DNA (54), and minimizing (by a factor of 25-fold) the probability of dissociation by an internal RecQ monomer from the assembly, relative to one dissociating at an end of the protein cluster (55).…”

“…Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9,16,(24)(25)(26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27)(28)(29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.…”

Fine tuning of a DNA fork by the RecQ helicase

“…These different speeds are the result of assembly of varying numbers of RecQ helicases loosely associating at the DNA fork, sort of "DNA unwinding by committee." 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.…”