Abstract
RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.
Highlights
The transient opening of nucleic acid duplexes is a key step in many DNA and RNA metabolic processes
Recent studies have shown that RecQ helicases, in addition to promoting DNA unwinding, can catalyze the opposite reaction—the pairing of the partially unwound DNA duplexes
We provide an initial view of the threedimensional structure of the larger complex and show that this state is associated with DNA strand annealing, whereas the smaller form carries out DNA unwinding
Summary
The transient opening of nucleic acid duplexes is a key step in many DNA and RNA metabolic processes. A feature common to all the proposed unwinding mechanisms is the necessity for helicases to contain multiple DNA or RNA binding sites in order to unwind the duplex and translocate processively without dissociating from the nucleic acid lattice [4,5]. The determination of the active oligomeric structure of a helicase is the first step toward understanding the mechanism of DNA unwinding Some helicases, such as PcrA from Bacillus stearothermophilus, function as monomers, and an ‘‘inchworm’’ unwinding model has been proposed in which the enzyme possesses two nonidentical DNA binding sites that simultaneously bind singlestranded DNA (ssDNA) and double-stranded DNA (dsDNA) [6,7]. Several replicative helicases, such as the bacteriophage T4 gp and the SV40 large T antigen proteins, have been shown to assemble into ring-shaped hexamers that might encircle the DNA resulting in high processivity [10,11,12]
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