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.

Characterization of a cooperativity domain mutant Lys3 --> Ala (K3A) T4 gene 32 protein.

The N-terminal "B" domain of T4 gene 32 protein contains a Lys3-Arg4-Lys5 sequence that has been postulated to provide a major determinant for cooperative binding. In this report, the equilibrium binding properties of a Lys3 --> Ala substitution mutant of gp32 (K3A gp32) and described and compared to a set of substitution mutants of Arg4 previously described (Villemain, J. L., and Giedroc, D. P. (1993) Biochemistry 32, 11235-11246) and further characterized here. K3A gp32 exhibits binding behavior which mirrors that of R4Q gp32. Despite an 6-8-fold decrease in overall binding affinity (Kapp = Kint x omega) at pH 8.1, 0.20 M NaCl, 20 degrees C, the magnitude of the cooperativity parameter is at most 2-3-fold smaller than that of the wild-type protein. The magnitude of omega is independent of salt concentration and type over the range in [NaCl] from 0.125 to 0. 225 M and [NaF] between 0.20 and 0.32 M (log omega = 2.86 +/- 0.19). For comparison, log omega for wild-type gp32 is 2.91 (+/- 0.21) resolved at 0.275 M NaCl and 3.39 +/- 0.11 in [NaF] between 0.40 and 0.45 M. In contrast to omega, the [NaCl] dependence of Kapp is large and markedly nonlinear for both wild-type and K3A gp32s over a [NaCl] range extending from 0.05 M to 0.40 M NaCl. Modeling of the complete salt dependence of Kapp for wild-type, K3A, and R4T gp32s in NaCl and NaF with a simple ion-exchange model suggests that substitutions within the basic Lys3-Arg4-Lys5 sequence do not strongly modulate the net displacement of cations and anions upon poly(A) complex formation by gp32.

The N-terminal B-domain of T4 gene 32 protein modulates the lifetime of cooperatively bound Gp32-ss nucleic acid complexes.

The N-terminal basic or B-domain (residues 1-21) of bacteriophage T4 gene 32 protein (gp32) provides a major determinant for highly cooperative binding by gp32 to single-stranded (ss) nucleic acids at equilibrium. In order to gain mechanistic insight into N-terminal domain function, the kinetics of dissociation of wild-type and previously characterized B-domain substitution mutant gp32s (R4K, R4Q, and K3A) from the model ribohomopolymer, poly(A), have been investigated under solution conditions identical to those used for equilibrium studies [Villemain, J. L., & Giedroc, D. P. (1993) Biochemistry 32, 11235-11246; Villemain, J. L., & Giedroc, D. P. (1996) J. Biol. Chem. 271, 27623-27629]. The dissociation of cooperatively bound gp32-poly(A) complexes was induced by sodium chloride concentration jumps and monitored by an increase in tryptophan fluorescence upon dissociation of the protein from poly(A) using stopped-flow techniques. The apparent dissociation rate constant, kd(app), for all mutant proteins studied was found to depend strongly on the initial fractional saturation of poly(A) just as was found previously for wild-type gp32. This permitted application of Lohman's model for the irreversible dissociation of cooperatively bound gp32-nucleic acid complexes [Lohman, T. M. (1983) Biopolymers 22, 1697-1713] from which the molecular rate constant, ke, the rate of dissociation of a protein monomer from teh end of a gp32-ss nucleic acid complex or protein cluster, could be determined. From the [NaCl]-dependence of kd(app), ke determined at 0.45 M NaCl, pH 8.1, 20 degrees C, was found to be 62 +/- 23, 78 +/- 8, 328 +/- 36, and 384 +/- 34 s-1 for wild-type, R4K, K3A, and R4Q gp32s, respectively. With the exception of R4K gp32, we find a striking correlation between the relative magnitudes of ke and Kapp, suggesting that the molecular defect in the equilibrium binding properties of the N-terminal domain mutants resides in the increased rate at which gp32 monomers dissociate from singly contiguous binding sites at the ends of clusters. The bimolecular association rate constant measured for wild-type gp32 and a weakly binding B-domain mutant, R4T gp32, to poly(dT) was found to be nearly identical, further evidence that the primary defect is in the dissociation reaction. We conclude that the N-terminal domain strongly modulates the lifetime of cooperatively bound gp32-polynucleotide complexes. The mechanistic and functional implications of these findings are discussed.

Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein.

Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids. The N-terminal "B" or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where K(app) = K(int) omega, omega > or = 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (omega = 1). In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gln4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32). The qualitative ranking of binding affinities to poly(A) is wild-type > or = R4K > R4Q > R4T > R4G > gp32-B (gp32 lacking the first 21 amino acids). The occluded site size is n(app) = 7.5 +/- 0.5 for all gp32s. Resolution of K(int) and omega for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (v < or = 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 degrees C, and a NaCl concentration where K(app) was (2-4) x 10(6) M-1 for each gp32 on the ribohomopolymer poly(A). Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the [NaCl] dependence of K(app) to 0.20 M NaCl, 20 degrees C, pH 8.1. The magnitude of omega was then assumed not to vary with [NaCl] (shown for R4T gp32), allowing estimation of K(int) at 0.20 M NaCl. We find that R4K gp32 binds to poly(A) with an overall affinity (K(app)) which is 2-3-fold lower than wild-type gp32, while omega for each molecule seems indistinguishable (wild-type gp32, omega approximately 800-1300; R4K gp32, omega approximately 600-1200). Surprisingly, R4Q gp32 is characterized by an omega also not readily distinguishable from the wild-type and R4K proteins (omega approximately 800-4400), while K(app) is reduced about 10-fold. This mutant also shows a significantly reduced [NaCl] dependence of the binding to poly(A). R4T gp32 binds about 10-fold weaker than the Q mutant. It exhibits an omega ranging from 300 to 700 and a substantially reduced [NaCl] dependence (delta log K(int)/delta log [NaCl] = -1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both K(int) and omega terms.(ABSTRACT TRUNCATED AT 400 WORDS)