Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.     

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.     

Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.     

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.     

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.     

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.     

Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.     

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.     

Relationship between hexamerization and ssDNA binding affinity in the uvsY recombination protein of bacteriophage T4

In bacteriophage T4, homologous genetic recombination events are catalyzed by a presynaptic filament containing stoichiometric quantities of the T4 uvsX recombinase bound cooperatively to single-stranded DNA (ssDNA). The formation of this filament requires the displacement of cooperatively bound gp32 (the T4 ssDNA-binding protein) from the ssDNA, a thermodynamically unfavorable reaction. This displacement is mediated by the T4 uvsY protein (15.8 kDa, 137 amino acids), which interacts with both uvsX- and gp32-ssDNA complexes and modulates their properties. Previously, we showed that uvsY exists as a hexamer under physiological conditions and that uvsY hexamers bind noncooperatively but with high affinity to ssDNA. We also showed that a fusion protein containing the N-terminal 101 amino acid residues of uvsY lacks interactions with uvsX and gp32 but retains both weak ssDNA-binding activity and a residual ability to stimulate uvsX-catalyzed recombination functions. Here, we present quantitative data on the oligomeric structure and ssDNA-binding properties of a closely related fusion protein designated uvsY. Sedimentation velocity and equilibrium results establish that uvsY, unlike native uvsY, behaves as a monomer in solution (M(app) = 14.2 kDa, = 2.1). Like native uvsY, uvsY binds noncooperatively to an etheno-DNA (epsilonDNA) lattice with a binding site size of 4 nucleotides/monomer; however at physiological ionic strength, the association constant for uvsY-epsilonDNA is decreased 10(4)-fold relative to native uvsY. Nevertheless, the magnitude of the salt effect on the association constant (K) is essentially unchanged between uvsY and uvsY, indicating that disruption of the C-terminus does not disrupt the electrostatic ssDNA-binding determinants found within each protomer of uvsY. Instead, the large difference in ssDNA-binding affinities reflects the loss of hexamerization ability by uvsY, suggesting that a form of intrahexamer synergism or cooperativity between binding sites within the uvsY hexamer leads to its high observed affinity for ssDNA.     

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.     

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.     

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.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.     

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.     

Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions

Bacteriophage T4 gene 32 encodes a single-stranded DNA (ssDNA) binding protein (gp32) required for T4 DNA replication, recombination, and repair. Previous physicochemical studies on gp32 and other ssDNA binding proteins have suggested that binding may involve hydrophobic interactions that result from the close approach of several aromatic amino acid side chains with the nucleic acid bases. In the case of gp32, five tyrosines and two phenylalanines have previously been implicated in gp32.ssDNA complex formation. Site-directed mutagenesis of T4 gene 32 was employed to produce a set of eight gp32 mutant proteins, each of which encoded a single substitution at one of the eight tyrosine residues within gp32. The mutant gp32 proteins were then subjected to physicochemical analysis to evaluate the role of each tyrosine residue in gp32 structure and function. Oligonucleotide binding studies suggest that tyrosine residues 84, 99, 106, 115, and 186 each contribute from 0.3 to 0.7 kcal/mol to ssDNA binding, which corresponds to 3-7% of the overall binding energy for gp32.ssDNA complex formation. Replacement of tyrosine residues 73 and 92 appears to lead to large structural changes that may be the result of disrupting the zinc binding subdomain within gp32.