Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp41-gp59 complex

The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.     

Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.     

Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.

The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replication forks. The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand displacement DNA synthesis and is essential for helicase and primosome assembly during T4 recombination-dependent DNA replication transactions. Of central importance is the ability of gp59 to load the gp41 helicase onto ssDNA previously coated with cooperatively bound molecules of gp32, the T4 ssDNA binding protein. Gp59 heteroassociations with ssDNA, gp32, and gp41 all appear to be essential for this loading reaction. Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupying ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59-gp32 complexes with or without ssDNA are currently unknown. To better understand gp59-gp32 interactions, we performed fluorescence anisotropy and analytical ultracentrifugation experiments employing native or rhodamine-labeled gp59 species in combination with altered forms of gp32, allowing us to determine their binding parameters, shape parameters, and other hydrodynamic properties. Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-domain required for cooperative binding to ssDNA and for stable self-association, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability. Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer. In both cases, heterodimer formation is accompanied by a conformational change in gp59 which correlates with decreased gp59-DNA binding affinity. Hydrodynamic modeling suggests an asymmetric prolate ellipsoid shape for gp59, consistent with its X-ray crystallographic structure, and this asymmetry appears to increase upon binding of gp32 derivatives. Implications of our findings for the structure and function of gp59 and gp59-gp32 complexes in T4 replication are discussed.     

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.     

Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA. Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate.

The T4 gp59 protein is the major accessory protein of the phage's replicative DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by promoting its assembly onto single-stranded (ss) DNA covered with cooperatively bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this helicase loading mechanism. Here, we characterize the properties of gp59-gp32-ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occupy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 species lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA and (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domain of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organizes gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes. Our results support a model in which gp59-gp32 interactions are non-essential for the co-occupancy of both proteins on ssDNA but are essential for the formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.     

Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA

Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).     

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Mechanistic studies of the T4 DNA (gp41) replication helicase: functional interactions of the C-terminal Tails of the helicase subunits with the T4 (gp59) helicase loader protein

We compare the activities of the wild-type (gp41WT) and mutant (gp41delta C20) forms of the bacteriophage T4 replication helicase. In the gp41delta C20 mutant the helicase subunits have been genetically truncated to remove the 20 residue C-terminal tail peptide domains present in the wild-type enzyme. Here, we examine the interactions of these helicase forms with the T4 gp59 helicase loader and the gp32 single-stranded DNA binding proteins, both of which are physically and functionally coupled with the helicase in the T4 DNA replication complex. We show that the wild-type and mutant forms of the helicase do not differ in their ability to assemble into dimers and hexamers, nor in their interactions with gp61 (the T4 primase). However they do differ in their gp59-stimulated unwinding activities and in their abilities to translocate along a ssDNA strand that has been coated with gp32. We demonstrate that functional coupling between gp59 and gp41 involves direct interactions between the C-terminal tail peptides of the helicase subunits and the loading protein, and measure the energetics and kinetics of these interactions. This work helps to define a gp41-gp59 assembly pathway that involves an initial interaction between the C-terminal tails of the helicases and the gp59 loader proteins, followed by a conformational change of the helicase subunits that exposes new interaction surfaces, which can then be trapped by the gp59 protein. Our results suggest that the gp41-gp59 complex is then poised to bind ssDNA portions of the replication fork. We suggest that one of the important functions of gp59 may be to aid in the exposure of the ssDNA binding sites of the helicase subunits, which are otherwise masked and regulated by interactions with the helicase carboxy-terminal tail peptides.     

Site-directed mutations of T4 helicase loading protein (gp59) reveal multiple modes of DNA polymerase inhibition and the mechanism of unlocking by gp41 helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.     

PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.     

Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.     

Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo

Bacteriophage T4 initiates origin-dependent replication via an R-loop mechanism in vivo. During in vitro reactions, the phage-encoded gp59 stimulates loading of the replicative helicase, gp41, onto branched intermediates, including origin R-loops. However, although gp59 is essential for recombination-dependent replication from D-loops, it does not appear to be required for origin-dependent replication in vivo. In this study, we have analyzed the origin-replicative intermediates formed during infections that are deficient in gp59 and other phage replication proteins. During infections lacking gp59, the initial replication forks from two different T4 origins actively replicated both leading- and lagging-strands. However, the retrograde replication forks from both origins were abnormal in the gp59-deficient infections. The lagging-strand from the initial fork was elongated as a new leading-strand in the retrograde direction without lagging-strand synthesis, whereas in the wild-type, leading- and lagging-strand synthesis appeared to be coupled. These results imply that gp59 inhibits the polymerase holoenzyme in vivo until the helicase-primase (gp41-gp61) complex is loaded, and we thereby refer to gp59 as a gatekeeper. We also found that all origin-replicative intermediates were absent in infections deficient in the helicase gp41 or the single-strand-binding protein gp32, regardless of whether gp59 was present or absent. These results argue that replication from the origin in vivo is dependent on both the helicase and single-strand-binding protein and demonstrate that the strong replication defect of gene 41 and 32 single mutants is not caused by gp59 inhibition of the polymerase.     

Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.     

Bacteriophage T4 32 protein is required for helicase-dependent leading strand synthesis when the helicase is loaded by the T4 59 helicase-loading protein

In the bacteriophage T4 DNA replication system, T4 gene 59 protein binds preferentially to fork DNA and accelerates the loading of the T4 41 helicase. 59 protein also binds the T4 32 single-stranded DNA-binding protein that coats the lagging strand template. Here we explore the function of the strong affinity between the 32 and 59 proteins at the replication fork. We show that, in contrast to the 59 helicase loader, 32 protein does not bind forked DNA more tightly than linear DNA. 32 protein displays a strong binding polarity on fork DNA, binding with much higher affinity to the 5' single-stranded lagging strand template arm of a model fork, than to the 3' single-stranded leading strand arm. 59 protein promotes the binding of 32 protein on forks too short for cooperative binding by 32 protein. We show that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein. However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein. Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein. Because 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein.     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.     

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.     

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.     

Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork

Processive strand-displacement DNA synthesis with the T4 replication system requires functional "coupling" between the DNA polymerase (gp43) and the helicase (gp41). To define the physical basis of this functional coupling, we have used analytical ultracentrifugation to show that gp43 is a monomeric species at physiological protein concentrations and that gp41 and gp43 do not physically interact in the absence of DNA, suggesting that the functional coupling between gp41 and gp43 depends significantly on interactions modulated by the replication fork DNA. Results from strand-displacement DNA synthesis show that a minimal gp41-gp43 replication complex can perform strand-displacement synthesis at approximately 90 nts/s in a solution containing poly(ethylene glycol) to drive helicase loading. In contrast, neither the Klenow fragment of Escherichia coli DNA polymerase I nor the T7 DNA polymerase, both of which are nonprocessive polymerases, can carry out strand-displacement DNA synthesis with gp41, suggesting that the functional helicase-polymerase coupling may require the homologous system. However, we show that a heterologous helicase-polymerase pair can work if the polymerase is processive. Strand-displacement DNA synthesis using the gp41 helicase with the T4 DNA polymerase holoenzyme or the phage T7 DNA polymerase-thioredoxin complex, both of which are processive, proceeds at the rate of approximately 250 nts/s. However, replication fork assembly is less efficient with the heterologous helicase-polymerase pair. Therefore, a processive (homologous or heterologous) "trailing" DNA polymerase is sufficient to improve gp41 processivity and unwinding activity in the elongation stage of the helicase reaction, and specific T4 helicase-polymerase coupling becomes significant only in the assembly (or initiation) stage.     

DNA Unwinding by Ring-Shaped T4 Helicase gp41 Is Hindered by Tension on the Occluded Strand

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, ‘occluded’ strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.     

A role for replication repair in the genesis of templated mutations

Replication repair mediates error-free bypass of DNA damage in a series of steps that include regression of the replication fork, primer-terminus switching to use the other daughter strand as an undamaged template, primer extension, primer switching back to its cognate template with the primer terminus now having bypassed the damage, and fork rearrangement to a normal configuration. By both genetic and biochemical criteria, bacteriophage T4 catalyzes replication repair with two alternative sets of proteins, one including the gp32 SSB and the gp41 DNA helicase and the other including the UvsX recombinase. In each pathway, synthesis is conducted by the gp43 DNA polymerase. Here we show that defects in gp32, gp41 or UvsX that impair replication repair also increase mutation rates generally, but especially for templated mutations. Such templated mutations are associated with palindromic or direct repeats that are either perfect or imperfect. Models of templated mutagenesis require that the primer terminus switches to an ectopic template, but one that yields mutations instead of error-free bypass. We suggest that the proteins that conduct replication repair normally direct a blocked primer strand specifically to the other daughter strand with considerable accuracy, but that strand switching becomes promiscuous when these proteins are mutationally impaired, thus promoting templated mutations.