In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro. We present evidence of a unique protein conformational change in the RecG helicase from an α-helix to a β-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, the α-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na+ and Mg2+. Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that the α-helix and β-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively. 1. Introduction The double-stranded conformation of genomic DNA must be unwound to provide single-stranded DNA (ssDNA) intermediates required for DNA replication, recombination, and repair. The ssDNA intermediates can adopt various structures like junctions, G-quadruplex, and intramolecular triplex [1–3]. In cells, the unwinding of double-stranded DNA (dsDNA) is catalyzed by a class of ubiquitous enzymes termed DNA helicases [4]. Helicases disrupt one or more base pairs within the duplex DNA and then translocate vectorially to the next duplex region to repeat the process [5–9]. The helicase activity is cycled by the binding and hydrolysis of an NTP through a number of energetic (conformational) states that have different affinities for ssDNA and dsDNA [10]. The structure of helicases plays a critical role in their catalytic functions. Previously, it was reported that almost all helicases appear to function as oligomers (usually dimers or hexamers) [10]. Oligomerization provides multiple binding sites necessary for DNA or RNA target recognition, interaction with accessory proteins, and ATP binding [5, 6]. RecG is a well-characterized helicase from Escherichia coli that unwinds DNA junctions in vitro. Biochemical studies revealed that RecG is active as a monomer [11]. It catalyzes the interconversion of forks and junctions [1, 12, 13]. It is necessary in cellular processes such as DNA replication, recombination, and repair [5, 6]. The conversion of a replication fork into a Holliday junction requires the simultaneous unwinding of the leading and lagging strands followed by the reannealing of the two parental strands and the annealing of the two nascent
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