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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumDefining the roles of individual residues in the single-stranded DNA binding site of PcrA helicaseMark S.Dillingham*†‡, Panos Soultanas*‡§, Paul Wiley*‡, Martin R.Webb¶, and Dale B.Wigley*||** *Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom; and ¶National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself. DNA-protein interactions | motor proteins | DNA translocation | mutagenesis The DNA helicases are a large and diverse group of motor proteins that are involved in the modulation of DNA structure. Frequently, they are found to catalyze the separation of duplex DNA into its component single strands (for reviews see refs. 1 and 2) but also are involved in events such as processing stalled replication forks and Holliday junction migration (3). These reactions are essential during DNA replication, recombination, and repair. Consequently, DNA helicases are found to be ubiquitous in nature, and organisms encode multiple DNA helicases that play specific cellular roles (4). PcrA is a superfamily I DNA helicase, found in Gram-positive bacteria, which is involved in DNA repair and plasmid rolling circle replication (5, 6). Its primary structure is very similar to the intensively studied Rep, UvrD and RecB(CD) helicases of Escherichia coli. PcrA from Bacillus stearothermophilus has been crystallized alone and in a variety of complexes with DNA and/or nucleotide analogues (7–9), and these structures have been used to devise a detailed molecular mechanism for PcrA-catalyzed helicase activity (8, 10). In this model, PcrA helicase is functional as a monomer that separates duplex DNA in a processive manner by coupling two distinct activities. First, PcrA translocates unidirectionally along single-stranded DNA (ssDNA) by virtue of a ssDNA tracking motor that is powered by ATP hydrolysis. Progression into duplex DNA is facilitated by a duplex destabilization activity acting ahead of the tracking motor, which also is modulated by ATP binding and hydrolysis. The crystal structures allow identification of the amino acid residues involved in binding ssDNA and proposed to be involved in the ssDNA tracking activity. They form a channel in which several bases of ssDNA bind in pockets formed predominantly by aromatic stacking interactions (Fig. 1). It is proposed that coordinated rearrangement of the relative positions of these residues in an ATP-dependent manner results in the ssDNA being passed unidirectionally along the base-stacking pockets (8). In the model, the motor is fueled by the hydrolysis of one ATP molecule for every base of ssDNA that is pumped through the protein (8). ssDNA translocation has been detected indirectly by studying Pi release from PcrA·DNA complexes, confirming the view that one ATP hydrolysis event is required for each base of movement along the DNA lattice (11). The residues that form the ssDNA binding channel are predicted by the model to be essential to helicase function. Many of the residues that contact DNA are found in the conserved helicase motifs that are characteristic of the superfamily I DNA helicases. Y257, W259, and R260 all are found within helicase motif III, which has been shown to play a critical role in coupling ATP hydrolysis to helicase activity (12–14). F64 is located in motif Ia, and H587 is found in the region between the closely aligned motifs V and VI. F192 and F626 are outside of the signature motifs but are conserved within the Rep/UvrD/PcrA family of SF1 DNA helicases (15). Here we present the results of a mutagenesis study of the residues that form the ssDNA binding site. The results of mutating the W259 and R260 residues to alanine have been reported previously and are consistent with a role in ssDNA binding and/or translocation (12). Both mutations result in helicase and DNA-binding defects, whereas kinetic parameters for ATP hydrolysis are close to wild-type values. In this study we have created alanine mutants for all of the remaining residues in the ssDNA binding site of PcrA helicase (F64A, F192A, Y257A, H587A, F626A). The mutant proteins have been studied biochemically to determine their ability to separate duplex DNA, hydrolyze ATP, bind ssDNA and double-stranded DNA (dsDNA), and translocate on ssDNA. The results confirm the essential nature of all of the residues for helicase activity. They also clarify the contribution of individual residues to the ssDNA translocation activity and illuminate the mechanism by which it is coupled to ATP hydrolysis. Materials and MethodsSite-Directed Mutagenesis. Site-directed mutagenesis of the pcrA gene was performed by using a PCR technique as described (16). This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.
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