Fine tuning of a DNA fork by the RecQ helicase.

Helicases are enzymes that couple the hydrolysis of ATP to the unwinding of duplex nucleic acids (NAs), thus providing the single-stranded NA (ssNA) intermediates necessary for nucleic acid processing and maintenance such as replication, recombination, repair, and transcription (1–3). All helicases possess a core helicase domain containing RecA-like motifs that are responsible for NA binding and ATP hydrolysis. Variation among individual helicases and helicase families within both the helicase core and in accessory domains allows this single class of enzymes to perform a myriad of different functions within the cell in different manners on various substrate types, resulting in helicases specific for every process involving NA in the cell. In PNAS, Rad et al. (4) provide a close-up view of a member of the RecQ family of enzymes that can modulate its activity to “fine tune” unwinding at a DNA fork. When DNA needs to be unwound, a helicase is typically involved. Unwinding produces a DNA fork structure, but the number of base pairs melted and the rate of melting are modulated to fit the needed task. For example, DNA replication requires a highly processive helicase activity, whereas some forms of DNA repair might require only a few base pairs to be melted. DNA recombination can involve unwinding of a few base pairs, or thousands of base pairs. Some helicases, such as the hexameric replicative helicases, are highly processive, unwinding kilobases of DNA at a time (5). However, many of the nonhexameric helicases in superfamilies 1 and 2 are typically nonprocessive, often unwinding only a few base pairs in a single binding event (6). Some notable exceptions to this are the superfamily 1 helicases RecBCD, which unwinds 30 kb on average before dissociation (7), and TraI, which can unwind hundreds of base pairs as a monomer (8). The RecQ family of helicases is involved in recombination, repair, and replication in both prokaryotes and eukaryotes (9). Humans have five RecQ helicases: RECQL1, BLM, WRN, RECQL4, and RECQL5. Defects in three of these, BLM, WRN, and RECQL4, are associated with diseases characterized by premature aging and cancer (10). The RecQ family has many biological roles. Escherichia coli RecQ is an unusual helicase in that it can unwind closed circular DNA (11), whereas most helicases require an ssDNA tail or sometimes can initiate unwinding at a dsDNA end. RecQ can both initiate homologous recombination and disrupt joint molecules by branch migration (12). Additionally, in conjunction with topoisomerase III, RecQ unwinds and promotes catenation of closed circular dsDNA (13) and resolves converging replication forks (14). Visualization of DNA unwinding at the single-molecule level has been accomplished using a number of systems in which total internal reflection fluorescence microscopy is applied. In single-molecule FRET (smFRET), the DNA substrate is typically visualized by labeling oligonucleotides with fluorescent probes such that appropriate FRET measurements can be made, leading to mechanistic conclusions. More recently, the proteins involved in the DNA unwinding process have been labeled with fluorescent probes to directly visualize events associated with protein binding and/or movement along the DNA. Such methods have provided stunning images and movies that greatly enable discernment of helicase mechanism(s). Rad et al. (4) developed an elegant system that allowed them to directly visualize the appearance of ssDNA during the unwinding process. They took advantage of the fact that the ssDNA binding protein (SSB) will associate with the newly created ssDNA after unwinding at the fork by RecQ. By labeling the SSB with fluorescent probes, unwinding forks appeared as fluorescent spots of varying intensity due to the accumulation of fluorescently labeled SSB. This approach allowed an important question to be addressed: How can the appropriate kinetic outcome for DNA unwinding be matched with a specific biological function? In the case of RecQ, the answer seems to depend on cooperative protein interactions. Quantitative analysis of the protein concentration dependence for rates of unwinding individual DNA forks revealed that varying numbers of RecQ molecules participate in different unwinding events. The results provide an avenue for interpreting how a helicase can fulfill diverse roles by assembly, with different kinetic outcomes dependent on the number of proteins bound at the DNA fork (Fig. 1).