The Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W has been used as a model system to identify genes that are required for growth at low temperature. Transposon mutagenesis was carried out to isolate mutant(s) of the bacterium that are defective for growth at 4Њ but normal at 22Њ. In one such cold-sensitive mutant (CS1), the transposon-disrupted gene was identified to be a homolog of the recD gene of several bacteria. Trans-complementation and freshly targeted gene disruption studies reconfirmed that the inactivation of the recD gene leads to a cold-sensitive phenotype. We cloned, sequenced, and analyzed ‫2.11ف‬ kbp of DNA from recD and its flanking region from the bacterium. recD was the last gene of a putative recCBD operon. The RecD ORF was 694 amino acids long and 40% identical (52% similar) to the Escherichia coli protein, and it could complement the E. coli recD mutation. The recD gene of E. coli, however, could not complement the cold-sensitive phenotype of the CS1 mutant. Interestingly, the CS1 strain showed greater sensitivity toward the DNA-damaging agents, mitomycin C and UV. The inactivation of recD in P. syringae also led to cell death and accumulation of DNA fragments of ‫03-52ف‬ kbp in size at low temperature (4Њ). We propose that during growth at a very low temperature the Antarctic P. syringae is subjected to DNA damage, which requires direct participation of a unique RecD function. Additional results suggest that a truncated recD encoding the N-terminal segment of (1-576) amino acids is sufficient to support growth of P. syringae at low temperature.

The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.beta hairpin | DNA replication | leading-strand synthesis | oligomerization | sliding helicase T he replisome of bacteriophage T7 can be reconstituted using T7 gp5 (DNA polymerase), Escherichia coli thioredoxin (trx) (processivity factor), T7 gp2.5 (ssDNA-binding protein) and T7 gp4 (helicase-primase) (1). The helicase domain of gp4 places it within the SF4 family of helicases (2). Like other members of this family, gp4 assembles onto ssDNA as a hexamer, an oligomerization that is facilitated by dTTP (3, 4). The inherent dTTPase of gp4 is stimulated approximately 40-fold by ssDNA, a stimulation that is required for its unidirectional translocation on ssDNA (4, 5). However, the molecular mechanism by which ssDNA enhances the hydrolysis of dTTP is not well understood. Although gp4 alone translocates on ssDNA and catalyzes limited unwinding of dsDNA, its major role in replication is manifest when it functions with T7 DNA polymerase within the replisome (1, 6). DNA synthesis activity of gp5/trx provides the driving force to accelerate DNA unwinding by gp4 (7). The association of gp5/trx with gp4 during leading-strand DNA synthesis increases the processivity for the 800 nucleotides observed using ssDNA templates to greater than 17 kb per binding event (1).The crystal structure of the hexameric gp4 shows a 6-fold symmetric ring with a central core of 25-30 Å, dimensions that resemble the size and shape of the gp4 hexameric rings seen in electron micrographs (3,8). Electron microscopy combined with other studies provides strong evidence that the ssDNA passes through the central core that provides the DNA-binding site. It is proposed that ssDNA transfers from one subunit to the adjacent subunit sequentially (8, 9). The crystal structure of gp4 also revealed the presence of three loops (loops I, II, and III) that protrude into the central cavity (8). Earlier mutational data had suggested that residues ar...

Background:The T7 DNA helicase couples the hydrolysis of dTTP to translocation on ssDNA and the unwinding of dsDNA. Results: Phe 523 , positioned in a ␤-hairpin loop at the subunit interface, plays a role in coupling the hydrolysis of dTTP to DNA unwinding. Conclusion: Phe 523 contacts the displaced complementary strand and facilitates unwinding. Significance: The mechanism of DNA unwinding by T7 DNA helicase.

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1-3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA) 3 as a hexamer and translocates 5Ј to 3Ј along the DNA strand using the energy of hydrolysis of dTTP (5-7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in v...

RecD is a 5¢ fi 3¢ helicase motor protein. The primary sequence contains the characteristic seven conserved motifs (I, Ia, II, III, IV, V, and VI) of the superfamily 1 (SF1) group of DNA helicases [1] (Fig. 1). In Escherichia coli, RecD displays ssDNA-dependent ATPase and helicase activity in vitro [2,3]. In vivo, it functions as a component of the RecBCD complex (also known as exonuclease V) that is involved in DNA repair and recombination in many bacteria [4]. RecBCD is a highly processive helicase ⁄ nuclease enzyme with dual motor activity, in which RecB and RecD subunits, with their respective (3¢ fi 5¢) and (5¢ fi 3¢) polar movement, translocate the enzyme along the anti-parallel strands of dsDNA. DNA unwinding by helicase activity is accompanied by degradation of the strands until the enzyme encounters the recombination hotspot v (chi) sequence (5¢-GCTGGTGG-3¢). This changes the nuclease property of the enzyme, leading to the generation of 3¢-extended ssDNA and loading of RecA onto the DNA for homologous pairing and DNA strand exchange, producing recombination intermediates [5]. Interestingly, however, RecBC alone is proficient for recombination and repair of DNA, and recD-inactivated mutants of E. coli do not show any growth defects [6,7]. Thus, the contribution of the RecD subunit is thought to be of less significance in vivo. Remarkably, RecD inactivation leads to the loss of exonuclease V activity in cells, despite the fact that the only nuclease catalytic center of RecBCD complex lies in the RecB subunit [8]. Hence, a role for RecD in regulating the nuclease activity of RecBCD has been advocated. Recently, using ATP RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5¢ overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNAbinding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4°C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.Abbreviations ABM, Antarctic bacterial medium; ATPc-S, adenosine 5¢-O-(thiophosphate); EMSA, electrophoretic gel mobility shift assay; SF1, superf...

The DNA helicase encoded by gene 4 of bacteriophage T7 forms a hexameric ring in the presence of dTTP, allowing it to bind DNA in its central core. The oligomerization also creates nucleotide-binding sites located at the interfaces of the subunits. DNA binding stimulates the hydrolysis of dTTP but the mechanism for this two-step control is not clear. We have identified a glutamate switch, analogous to the glutamate switch found in AAA؉ enzymes that couples dTTP hydrolysis to DNA binding. A crystal structure of T7 helicase shows that a glutamate residue (Glu-343), located at the subunit interface, is positioned to catalyze a nucleophilic attack on the ␥-phosphate of a bound nucleoside 5 -triphosphate. However, in the absence of a nucleotide, Glu-343 changes orientation, interacting with Arg-493 on the adjacent subunit. This interaction interrupts the interaction of Arg-493 with Asn-468 of the central ␤-hairpin, which in turn disrupts DNA binding. When Glu-343 is replaced with glutamine the altered helicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. When both Arg-493 and Asn-468 are replaced with alanine, dTTP hydrolysis is no longer stimulated in the presence of DNA. Taken together, these results suggest that the orientation of Glu-343 plays a key role in coupling nucleotide hydrolysis to the binding of DNA.Helicases are molecular motors that translocate unidirectionally along single-stranded nucleic acids using energy derived from nucleotide hydrolysis (1-3). The gene 4 protein (gp4) 2 encoded by bacteriophage T7 is a bifunctional protein that contains both a helicase and primase domain. The helicase domain, located in the C-terminal half of the protein, is tethered to the primase domain, located in the N-terminal half, by a flexible linker. In the presence of dTTP gp4 oligomerizes to form hexamers and heptamers, a process involving the helicase domain and linker region. Gp4 binds, as a hexamer, to singlestranded DNA (ssDNA) in the presence of dTTP and translocates in a 5Ј to 3Ј direction along the DNA strand using the energy derived from the hydrolysis of dTTP (4 -6). The ssDNA passes through the center of the hexamer and binding of residues within the central core to the DNA stimulates nucleotide hydrolysis, a phenomenon also observed for other members of the SF IV group of helicases (3). The nucleotide-binding sites are located at the interfaces between the subunits with dTTP being the preferred nucleotide for unwinding of duplex DNA (7). Upon binding of dTTP, the helicase recruits ssDNA into its central core (8 -11).Like the SF IV group of helicases, AAAϩ ATPases (ATPases associated with diverse cellular activities) also form oligomeric structures (often hexamers) that form a ring-shaped structure with a central pore. These proteins function as a molecular motor that couples ATP binding and hydrolysis to changes in conformational states. These conformational changes are propagated through the assembly to enable the protein to bind to its substrate, to translocate on it, and to remodel...

The functional form of the DNA helicase encoded by bacteriophage T7 is a hexamer of six identical subunits. The helicase couples the hydrolysis of dTTP to unidirectional translocation and unwinding of DNA. The nucleotide binding site of the helicase is located at the subunit interface. In a crystallographic structure of the T7 helicase with bound nucleoside 5′‐triphosphate, Phe523 positioned in a beta‐hairpin loop at the sub‐unit interface is buried within the interface. However, in the unbound state, Phe523 is more exposed towards the outer surface of the helicase. This different orientation suggests that Phe523 and the associated beta‐hairpin loop may undergo a conformational change coupled with nucleotide hydrolysis. Replacement of Phe523 with alanine or valine abolishes the ability of the helicase to unwind DNA and to stimulate T7 polymerase activity on double‐stranded DNA. Genetic and biochemical experiments indicate that a hydrophobic residue with longer side chains is essential at this position. In addition, Phe523 appears to play a critical role in coupling the hydrolysis of dTTP to DNA unwinding. We postulate that upon dTTP hydrolysis, Phe523 moves from within the sub‐unit interface to a more exposed position where it may intercalate into double stranded DNA to facilitate DNA unwinding by the helicase. Results from this study support the strand exclusion mechanism of DNA unwinding by ring helicases.